Methods and compositions for modulating the immune system with Arginase I

ABSTRACT

Methods and compositions comprising recombinant Arginase I proteins which are capable of depleting the plasma arginine levels in a subject are disclosed. The methods and compositions can be used to modulate the activity of the immune system in a subject. Modulation of the immune system is useful in the treatment of immune disorders and in preventing rejection of a transplanted organ, tissue, or cell. The methods and compositions can also be used to treat a bone condition of a subject.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.14/697,835, filed on Apr. 28, 2015, which claims the benefit of U.S.Provisional Patent Application Ser. No. 61/985,924, filed on Apr. 29,2014, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety, Said ASCII copy, created on Jul. 16, 2015, isnamed 46106-701.201_SL/txt and is 50,705 bytes in size.

BACKGROUND OF THE INVENTION

The immune system functions to protect the body against harmfulantigens, bacteria, viruses, toxins, blood or tissues from anotherperson or species, and cancer cells. Immune system disorders can lead tohyperactivity or hypoactivity of the immune system. In cases of immunesystem hyperactivity, the body attacks and damages its own tissues. Incases of immune system hypoactivity, also known as immune deficiency,the body's ability to fight foreign antigens is diminished, which oftenleads to a greater vulnerability to infections.

The enzyme arginase metabolizes L-arginine to L-ornithine and urea.Besides its fundamental role in the hepatic urea cycle, arginase isexpressed in some cells of the immune system of some mammals. However,it is unclear if interferences with L-arginine metabolism can be used totreat immune conditions. Importantly, several challenges exist informulating exogenous Arginases as a therapeutic agent that is suitablefor clinical administration.

SUMMARY OF THE INVENTION

In some embodiments, the invention provides a method of treating aninflammatory disease in a subject in need thereof, the method comprisingadministering to the subject a therapeutically-effective amount of apurified Arginase or a functional fragment thereof.

In some embodiments, the invention provides a method of modulatinginflammation, the method comprising administering to a subject atherapeutically-effective amount of a purified Arginase, or a functionalfragment thereof, wherein the administration modulates the inflammation.

In some embodiments, the purified Arginase is recombinant Arginase. Insome embodiments, the recombinant Arginase is pegylated. In someembodiments, a functional fragment of the recombinant Arginase ispegylated. In some embodiments, the pegylated recombinant Arginase isrecombinant human Arginase I, or a functional fragment thereof.

In some embodiments, the invention provides a method of modulatinginflammation, the method comprising administering to a subject atherapeutically-effective amount of a purified pegylated recombinanthuman Arginase I, or a functional fragment thereof, wherein theadministration modulates the inflammation.

In some embodiments, the invention provides a use of a purifiedrecombinant arginase, or a functional fragment thereof, in thepreparation of a medicament for treating an inflammatory disease in asubject.

In some embodiments, the invention provides a pharmaceutical compositioncomprising a purified pegylated recombinant human Arginase I protein, ora functional fragment thereof, and at least one polyethylene glycololigomer. In some embodiments, the pegylated recombinant human ArginaseI protein comprises at least two polyethylene glycol oligomers, whereineach polyethylene glycol oligomer weighs from about 20 kilodaltons toabout 40 kilodaltons. In some embodiments the pegylated recombinantArginase I protein, or a functional fragment thereof, comprises fromabout 4 to about 13 polyethylene glycol oligomers, wherein eachpolyethylene glycol oligomer weighs about 5 kilodaltons.

In some embodiments, the purified pegylated recombinant human ArginaseI, or a functional fragment thereof, modulates inflammation byinhibiting T-cell polarization. In some embodiments, the purifiedpegylated recombinant human Arginase I, or a functional fragmentthereof, inhibits T-cell polarization by modulating cytokine release. Insome embodiments, the purified pegylated recombinant human Arginase I,or a functional fragment thereof, modulates expression of Interleukin 6(IL-6). In some embodiments, the purified pegylated recombinant humanArginase I, or a functional fragment thereof, modulates expression ofInterferon gamma (INFγ). In some embodiments, the administration of thepurified pegylated recombinant human Arginase I, or a functionalfragment thereof, depletes the level of arginine in the plasma of asubject to below 10 μM.

In some embodiments, the pharmaceutical composition and method provide amethod for treating an autoimmune disorder. In some embodiments, theautoimmune disorder is multiple sclerosis. In some embodiments, theautoimmune disorder is rheumatoid arthritis.

In some embodiments, the disclosure provides a method of treating a bonecondition in a subject in need thereof, the method comprisingadministering to the subject a therapeutically-effective amount of apurified Arginase, or functional fragment thereof. In some cases, thebone condition is osteoporosis. In other cases, the bone condition isinflammation.

In some embodiments, the purified recombinant human Arginase I is SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, orSEQ ID NO: 16. In some embodiments, the recombinant human Arginase I ispegylated.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the upregulation of Arginase I by LPS in macrophages.

FIG. 2 illustrates an increase in Arginase I expression accompanied byloss of PTEN.

FIG. 3 illustrates the function of C/EBPβ in PTEN deficient macrophages.

FIG. 4 illustrates that constitutive activation of PI3K promotesArginase I expression and release into the extracellular space.

FIG. 5 illustrates inhibition of T-cell polarization by Arginase I.

FIG. 6 illustrates results of a treatment of an art recognized model ofmultiple sclerosis, the experimental autoimmune encephalomyelitis (EAE)mouse, with recombinant human Arginase I.

FIG. 7 depicts graphs measuring various clinical parameters of arthriticmice treated with recombinant human Arginase I.

FIG. 8 illustrates the reduction of paw swelling in arthritic micetreated with recombinant human Arginase I.

FIG. 9 illustrates the reduction of systemic release of Interleukin 6(IL-6) in arthritic mice treated with human recombinant human ArginaseI.

FIG. 10 depicts graphs measuring the expression of pro-inflammatorycytokines post collagen immunization.

FIG. 11 is a graph depicting the clinical score of a mouse model ofexperimental autoimmune encephalomyelitis (EAE) treated with recombinanthuman Arginase I.

FIG. 12 depicts the fluorescence-activated cell sorting analysis ofpopulations of immune cells in EAE mice treated with recombinant humanArginase I.

FIG. 13 depicts the results of fluorescence-activated cell sortingexperiments indicating that treatment with recombinant human Arginase Iprevents T-cell proliferation.

FIG. 14 is a schematic of a process utilized for optimizing apharmaceutical composition comprising a purified Arginase I.

FIG. 15 is a graph illustrating the serum arginine depletion by apurified recombinant human Arginase I pegylated with mPEG-MAL (—SHmodification).

FIG. 16 is a graph illustrating the serum arginine depletion withvarious purified recombinant human Arginase(s) pegylated on Lysresidues.

FIG. 17 is a graph illustrating the degree of pegylation of variouspurified recombinant human Arginase I proteins by amine (—NH₂)conjugation.

FIG. 18 is a graph illustrating the epitope analysis of a purifiedpegylated recombinant human Arginase I.

FIG. 19 illustrates IFNγ and IL-17A mRNA expression levels from myelinoligodendrocyte glycoprotein (MOG) restimulated T-cells inhibited withpurified human Arginase I.

FIG. 20 illustrates IFNγ and IL-17A protein expression levels frommyelin oligodendrocyte glycoprotein (MOG) restimulated T-cells inhibitedwith purified human Arginase I.

FIG. 21 illustrates improvements in clinical score of experimentalautoimmune encephalomyelitis (EAE) mice treated with a recombinant humanArginase I.

FIG. 22 is a schematic of an osteoclast differentiation assay.

FIG. 23 is a graph illustrating an osteoclast assay of differentiatedwildtype bone marrow derived macrophages treated with a recombinanthuman Arginase I.

FIG. 24 is a graph demonstrating that expression of Arginase I can belost during osteoclastogenesis.

FIG. 25 illustrates that addition of recombinant Arginase I duringosteoclast differentiation can modulate osteoclast formation.

FIG. 26 is a graph illustrating that blockage of osteoclastogenesis canbe dependent on the catalytic functions of recombinant human Arginase I(recArgI).

FIG. 27 is a graph illustrating an assay where addition of recombinantArginase I to hematopoietic stem cells did not influence osteoclastformation.

FIG. 28 is a graph illustrating the effects of different dosages ofrecombinant human Arginase I (recArgI) on day 0 or on day 6osteoclastogenesis.

FIG. 29 is a graph illustrating the mRNA expression levels ofosteoclastogenesis genes after 7 days of differentiation with andwithout incubation with recombinant human Arginase I (recArgI).

DETAILED DESCRIPTION OF THE INVENTION

Most living beings are exposed to a number of different antigens everyday. However some animals possess immune systems that are capable ofresponding to such antigens, and protecting against the initiation orformation of disease. To function properly, an immune system must detecta wide variety of antigens, such as virus(es), parasitic worm(s), orallergen(s) and initiate a response in the body against foreignsubstances, abnormal cells and/or tissues. In a response to an unknownantigen, a healthy immune system begins to produce antibodies.

In some diseases however, an immune system can start producingantibodies that instead of fighting infections, attack the body's owntissues. This can lead to autoimmune diseases. Cancerous growths,including malignant cancerous growths, can also be recognized by theinnate immune cells of a subject and trigger an immune response. Theactivation of innate immune cells triggers numerous intracellularsignaling pathways, which require tight control in order to mount anadequate immune response.

Arginase I is a key element of the urea cycle, which converts arginineto urea, and is predominately active in the liver. Arginase I also playa functional role in the immune system. T-cells for instance aredependent on the semi-essential amino acid arginine to mature andrespond to infections. Expression of Arginase I in innate immune cellsleads to depletion of arginine levels from a physiological system underinflammatory conditions. For example, Arginase I expression in myeloidcells can lead to T-cell anergy and prevent T-helper cell functions.

In mice, Arginase I is expressed by cells of monocytic origin. Inhumans, Arginase I is constitutively expressed ingranulocytes/neutrophils and participates in fungicidal activity.Arginase I expressing macrophages are considered by some to bealternatively activated or M2 macrophages, involved in tissueregeneration and repair but also in the immune defense againstmulticellular pathogens and parasites. Arginase I expression in murinemyeloid cells is regulated by Th2 cytokines IL-4/IL-13. However, it isunclear if human Arginase I and the murine Arginase I work by a similarmechanism of action.

The PI3K/PTEN signaling pathway plays a functional role in numerousphysiologically important processes such as innate immunity, cellsurvival, proliferation, migration and metabolism. ThePhosphatidylinositol-3 Kinase (PI3K) signaling pathway can downregulatethe expression and release of pro-inflammatory cytokines in some cells.These signaling processes are strictly regulated by the lipidphosphatase PTEN, an antagonist of the PI3K pathway. PTEN is a tumorsuppressor that is responsible for the elevated production of cytokinessuch as Interleukin 6 (IL-6) in response to Toll like receptor (TLR)agonists. PI3K activation is considered to be pro-inflammatory andmodulation of the PI3K pathway is indispensable for proper guidance ofimmune cells to the site of infection or inflammation.

We describe herein experiments characterizing the addition ofrecombinant pegylated Arginase I to cultured cells and mouse models. Theexperiments demonstrate that extracellular Arginase I can exert potentanti-inflammatory effects on immune cells. Transfer experiments ofconditioned media derived from naïve PTEN^(−/−) macrophages, containinghigh amounts of Arginase I, showed reduced expression ofpro-inflammatory T-cell polarizing cytokines in cultured cells andanimal models.

The invention disclosed herein provides compositions and methods fortreating conditions associated with the immune system by administratingrecombinant Arginase I proteins to a subject to modulate the PI3K/PTENsignaling pathway and cytokine secretion. In some embodiments, theinvention disclosed herein provides a method of modulating inflammationby administering to a subject a therapeutically-effective amount of apurified pegylated recombinant human Arginase I.

The disclosure demonstrates that a functional consequence of sustainedArginase I expression in a physiological system is the formation of ahypo-inflammatory environment by diminished function of T-cell mediatedpathophysiologic effects in vitro and in vivo. The finding provides arobust and effective method for the modulation of an immune system. Suchmodulation provides an effective treatment for a variety of immuneconditions, such as multiple sclerosis and rheumatoid arthritis. Inaddition, modulation of the immune system with a recombinant Arginase ofthe disclosure can be used alongside surgical procedures, for example,to provide a hypo-inflammatory environment that reduces the likelihoodof cell/tissue rejection during organ transplantation.

In some aspects, the disclosure provides a method of modulatinginflammation, the method comprising administering to a subject atherapeutically-effective amount of a purified pegylated recombinanthuman Arginase I, or a functional fragment thereof. In some cases, thepurified pegylated recombinant human Arginase I, or functional fragmentthereof, modulates inflammation by inhibiting T-cell polarization. Insome cases, the purified pegylated recombinant human Arginase I inhibitsT-cell polarization by modulating cytokine release.

Another aspect of the disclosure provides a pharmaceutical compositioncomprising a purified recombinant human Arginase I protein conjugated toat least one polyethylene glycol oligomer. In some cases, the at leastone polyethylene glycol oligomer is methoxy poly(ethylene glycol).

The disclosure also demonstrates a functional role for Arginases in bonephysiology. Bone formation is a multi-complex procedure that includesmany stages, and each one of them presents as a potential target fortherapeutic intervention. Inflammation can also interfere with theability of a vertebrate body to repair bone mass. In some aspects, thedisclosure demonstrates that expression of Arginase I is lost duringosteoclastogenesis, and addition of a recombinant Arginase I duringosteoclast differentiation can modulate at least osteoclastogenesis. Thedisclosure also demonstrates that blockage if osteoclastogenesis isdependent on the catalytic functions of recombinant Arginase I. Thefindings suggest that modulation of Arginase expression can provide aneffective treatment for a variety of bone conditions, includingosteoporosis. Modulation of Arginase I expression can also provide aneffective treatment for osteoporosis by reducing chronic inflammation inthe bone, which can be an aggravating factor in osteoporosis.

Methods of Treating Immune Disorders, Bone Conditions, and Cancers.

The methods, compositions, and kits of this disclosure may comprise amethod to treat, arrest, reverse, or ameliorate a disease. In somecases, the disease may be an autoimmune disease. In some cases, thedisease may be a bone condition, such as osteoporosis. In some cases,the modulation is achieved by administrating a therapeutically-effectivedose of a recombinant Arginase protein or a functional fragment thereof.In some cases, the protein is recombinant human Arginase I or afunctional fragment thereof.

Arginase I is an important modulator of the innate and adaptive immuneresponses. A plurality of subjects afflicted with immune systemdisorders and cancers can benefit from the use of a recombinant humanArginase I. Subjects can be humans, non-human primates such aschimpanzees, and other apes and monkey species; farm animals such ascattle, horses, sheep, goats, swine; domestic animals such as rabbits,dogs, and cats; laboratory animals including rodents, such as rats, miceand guinea pigs, and the like. A subject can be of any age. Subjects canbe, for example, elderly adults, adults, adolescents, pre-adolescents,children, toddlers, infants.

Also recognized herein is the therapeutic potential of a recombinantArginase protein in treating various bone conditions, such asosteoporosis or inflammation in the bones. The strength and integrity ofthe vertebrate skeleton, e.g., the human skeleton, depends on a delicateequilibrium between bone resorption by osteoclasts and bone formation byosteoblasts. In osteoporosis, this balance shifts in favor ofosteoclasts, and bone resorption exceeds bone formation. In some cases,a recombinant Arginase protein, or fragment thereof, can shift thebalance between osteoclast and osteoblast formation.

The activity of a plurality of cells in the immune system can bemodulated by a recombinant Arginase I. Non-limiting examples of cellswhose activity can be modulated by recombinant Arginase I include: Bcells; CD4; CD8; blood cells, including red blood cells and white bloodcells; dendritic cells, including dendritic antigen presenting cells;macrophages; memory B cells; memory T cells; monocytes; natural killercells; neutrophil granulocytes; T-helper cells; and T-killer cells. Theactivity of a plurality of additional cells can also be modulated by arecombinant Arginase I. Non-limiting examples of cells whose activitycan be modulated by recombinant arginase I include hematopoietic stemcells, osteoclasts, osteoblasts, osteoprogenitor, osteocytes, andprecursors or derivatives thereof.

Examples of immune diseases or conditions that can be treated with apurified Arginase disclosed herein include rheumatoid arthritis,multiple sclerosis, experimental autoimmune encephalomyelitis,psoriasis, uveitis, diabetes mellitus type 1, systemic lupuserythematosus (SLE), eczema, scleroderma, ulcerative proctitis, severecombined immunodeficiency (SCID), DiGeorge syndrome,ataxia-telangiectasia, seasonal allergies, perennial allergies, foodallergies, anaphylaxis, mastocytosis, allergic rhinitis, atopicdermatitis, Parkinson's, Alzheimer's, hypersplenism, leukocyte adhesiondeficiency, X-linked lymphoproliferative disease, X-linkedagammaglobulinemia, selective immunoglobulin A deficiency, hyper IgMsyndrome, HIV, autoimmune lymphoproliferative syndrome, Wiskott-Aldrichsyndrome, chronic granulomatous disease, common variableimmunodeficiency (CVID), hyperimmunoglobulin E syndrome, Hashimoto'sthyroiditis, acute inflammatory conditions, chronic inflammatoryconditions, and cancer.

In some embodiments, a bone condition can be treated with a purifiedArginase. Non-limiting examples of bone conditions include:osteoporosis, Paget's disease, osteogenesis imperfecta, fibrousdysplasis, or osteomyelitis. In some cases, the bone condition isassociated with a misregulation in osteoclast or osteoblast function.

In some embodiments, a cancer is susceptible to treatment with apurified Arginase. Non-limiting examples of cancers include: acutelymphoblastic leukemia, acute myeloid leukemia, adrenocorticalcarcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer,appendix cancer, astrocytomas, neuroblastoma, basal cell carcinoma, bileduct cancer, bladder cancer, bone cancers, brain tumors, such ascerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodermaltumors, visual pathway and hypothalamic glioma, breast cancer, bronchialadenomas, Burkitt lymphoma, carcinoma of unknown primary origin, centralnervous system lymphoma, cerebellar astrocytoma, cervical cancer,childhood cancers, chronic lymphocytic leukemia, chronic myelogenousleukemia, chronic myeloproliferative disorders, colon cancer, cutaneousT-cell lymphoma, desmoplastic small round cell tumor, endometrialcancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ celltumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoidtumor, gastrointestinal stromal tumor, gliomas, hairy cell leukemia,head and neck cancer, heart cancer, hepatocellular (liver) cancer,Hodgkin lymphoma, Hypopharyngeal cancer, intraocular melanoma, isletcell carcinoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip andoral cavity cancer, liposarcoma, liver cancer, lung cancers, such asnon-small cell and small cell lung cancer, lymphomas, leukemias,macroglobulinemia, malignant fibrous histiocytoma of bone/osteosarcoma,medulloblastoma, melanomas, mesothelioma, metastatic squamous neckcancer with occult primary, mouth cancer, multiple endocrine neoplasiasyndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavity andparanasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma,non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer,oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma ofbone, ovarian cancer, ovarian epithelial cancer, ovarian germ celltumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinusand nasal cavity cancer, parathyroid cancer, penile cancer, pharyngealcancer, pheochromocytoma, pineal astrocytoma, pineal germinoma,pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia,primary central nervous system lymphoma, prostate cancer, rectal cancer,renal cell carcinoma, renal pelvis and ureter transitional cell cancer,retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, skincancers, skin carcinoma merkel cell, small intestine cancer, soft tissuesarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma,throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastictumor (gestational), cancers of unknown primary site, urethral cancer,uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrommacroglobulinemia, and Wilms tumor.

The treatment may comprise treating a subject (e.g. a patient with adisease and/or a lab animal with a condition) with an Arginase of thedisclosure. The disease may be an autoimmune disease. The disease may bean inflammatory disease. The subject may be a human. Treatment may beprovided to the subject before clinical onset of disease. Treatment maybe provided to the subject after clinical onset of disease. Treatmentmay be provided to the subject after 1 day, 1 week, 6 months, 12 months,or 2 years after clinical onset of the disease. Treatment may beprovided to the subject for more than 1 day, 1 week, 1 month, 6 months,12 months, 2 years or more after clinical onset of disease. Treatmentmay be provided to the subject for less than 1 day, 1 week, 1 month, 6months, 12 months, or 2 years after clinical onset of the disease.Treatment may also include treating a human in a clinical trial. Atreatment can comprise administering to a subject a pharmaceuticalcomposition, such as one or more of the pharmaceutical compositionsdescribed throughout the disclosure. A treatment can comprise modulatingthe levels of endogenous arginine in vivo.

Mutant Recombinant Arginases.

Sustained expression of Arginase I proteins can be used clinically toprovide a hypo-inflammatory environment in vitro and in vivo (Furtherdescribed, for instance, in Example 1, and FIGS. 1-6). However, onechallenge encountered in formulating a purified Arginase for therapeuticpurposes is the formation of protein aggregates in solution due tointer-chain disulfide bond formation. To overcome some of the challengesin preparing a purified Arginase that is suitable for clinicaladministration, a series of mutations were performed in the sequence ofa wild-type human Arginase I. TABLE 1 describes various site-specificmutants of human Arginase I that have been designed to reduce theaggregation of a purified recombinant Arginase I in solution. TABLE 1also describes various human Arginase I sequences comprising a moleculartag (SEQ ID NO: 9-16).

TABLE 1 SEQ ID NO: Sequence SEQ ID NO:MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 1KEQECDVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPCIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LACFGLAREG NHKPIDYLNP PK SEQ ID NO:MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 2KEQECDVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPCIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LAAFGLAREG NHKPIDYLNP PK SEQ ID NO:MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 3KEQECDVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPAIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LACFGLAREG NHKPIDYLNP PK SEQ ID NO:MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 4KEQEADVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPCIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LACFGLAREG NHKPIDYLNP PK SEQ ID NO:MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 5KEQECDVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPAIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LAAFGLAREG NHKPIDYLNP PK SEQ ID NO:MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 6KEQEADVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPCIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LAAFGLAREG NHKPIDYLNP PK SEQ ID NO:MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 7KEQEADVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPAIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LACFGLAREG NHKPIDYLNP PK SEQ ID NO:MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 8KEQEADVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPAIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LAAFGLAREG NHKPIDYLNP PK SEQ ID NO:MHHHHHH MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 9KEQECDVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPCIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LACFGLAREG NHKPIDYLNP PK SEQ ID NO:MHHHHHH MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 10KEQECDVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPCIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LAAFGLAREG NHKPIDYLNP PK SEQ ID NO:MHHHHHH MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 11KEQECDVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPAIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LACFGLAREG NHKPIDYLNP PK SEQ ID NO:MHHHHHH MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 12KEQEADVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPCIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LACFGLAREG NHKPIDYLNP PK SEQ ID NO:MHHHHHH MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 13KEQECDVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPAIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LAAFGLAREG NHKPIDYLNP PK SEQ ID NO:MHHHHHH MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 14KEQEADVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPCIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LAAFGLAREG NHKPIDYLNP PK SEQ ID NO:MHHHHHH MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 15KEQEADVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPAIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LACFGLAREG NHKPIDYLNP PK SEQ ID NO:MHHHHHH MSAKSRTIGI IGAPFSKGQP RGGVEEGPTV LRKAGLLEKL 16KEQEADVKDY GDLPFADIPN DSPFQIVKNP RSVGKASEQLAGKVAEVKKN GRISLVLGGD HSLAIGSISG HARVHPDLGVIWVDAHTDIN TPLTTTSGNL HGQPVSFLLK ELKGKIPDVPGFSWVTPAIS AKDIVYIGLR DVDPGEHYIL KTLGIKYFSMTEVDRLGIGK VMEETLSYLL GRKKRPIHLS FDVDGLDPSFTPATGTPVVG GLTYREGLYI TEEIYKTGLL SGLDIMEVNPSLGKTPEEVT RTVNTAVAIT LAAFGLAREG NHKPIDYLNP PK

A mutant recombinant Arginase can comprise one or more mutations.Suitable amino acid modifications for improving the rheology of anArginase I can be conservative or non-conservative mutations. A mutationcan be made such that the encoded amino acid is modified to a polar,non-polar, basic or acidic amino acid. A recombinant Arginase of theinvention can be a wild type human Arginase. A recombinant Arginase ofthe invention can be a mutated human Arginase. A recombinant Arginase Ican be generated from recombinant DNA, for example with biomolecularengineering techniques. A purified Arginase I can be an arginase that isextracted from a crude extract, such as a whole cell lysate. A purifiedrecombinant Arginase I can be an Arginase I that is purified from, forexample, the crude extract of a biological system designed to expressthe recombinant Arginase I.

TABLE 1 discloses protein sequences of various mutant recombinant humanArginases I. SEQ ID NO: 1 corresponds to a wild-type human Arginase. SEQID NOs 2-8 are mutated sequences of SEQ ID NO: 1. SEQ ID NO: 2 comprisesa C303→A303 mutation. SEQ ID NO: 3 comprises a C168→A168 mutation. SEQID NO: 4 comprises a C45→A45 mutation. SEQ ID NO: 5 comprises theC303→A303 and C168→A168 double mutations. SEQ ID NO: 6 comprises theC303→A303 and C45→A45 double mutations. SEQ ID NO: 7 comprises theC168→A168 and C45→A45 double mutations. SEQ ID NO: 8 comprises theC303→A303, C168→A168, and C45→A45 triple mutations.

A recombinant human Arginase I can have a molecular tag engineered intothe recombinant nucleic acid sequence. A molecular tag can facilitatepurification of a recombinant Arginase from a crude expression system. Amolecular tag can be, for example, a polyhistidine tag, aglutathione-S-transferase (GST) tag, a maltose binding protein (MBP)tag, or a chitin binding protein (CBP) tag. In some embodiments, amolecular tag comprises a polyhistidine tag. A molecular tag can bepresent, for example, in the amino-terminus or in the carboxy terminusof a recombinant Arginase.

TABLE 1 also discloses protein sequences of various mutant recombinanthuman Arginases comprising a molecular tag. SEQ ID NO: 9 corresponds toa wild-type human Arginase comprising a polyhistidine tag. SEQ ID NOs:10-16 are mutated sequences of SEQ ID NO: 1 comprising a tag. SEQ ID NO:10 comprises a polyhistidine tag and a C303→A303 mutation. SEQ ID NO: 11comprises a polyhistidine tag and a C168→A168 mutation. SEQ ID NO: 12comprises a polyhistidine tag and a C45→A45 mutation. SEQ ID NO: 13comprises a polyhistidine tag, the C303→A303, and the C168→A168 doublemutations. SEQ ID NO: 14 comprises a polyhistidine tag, the C303→A303and the C45→A45 double mutations. SEQ ID NO: 15 comprises apolyhistidine tag, the C168→A168 and the C45→A45 double mutations. SEQID NO: 16 comprises a polyhistidine tag, the C303→A303, the C168→A168,and the C45→A45 triple mutations. In some cases, a therapeuticrecombinant human Arginase can be a functional fragment of an Arginasedescribed in TABLE 1.

A recombinant Arginase, or a functional fragment thereof, can beexpressed/produced, for example, in vivo from bacterial cells, insectcells, mammalian cells, synthetic cells, or in vitro from cell-freesystems or chemical synthesis. A recombinant Arginase I can be coded byany combination of codons in the degenerate code. In some embodiments,nucleotides are replaced by taking note of the genetic code such that acodon is changed to a different codon that codes for the same amino acidresidue. In some embodiments, altering the identity of a cysteineresidue as described in TABLE 1 can result in a reduction of proteinaggregation in solution of: about 2%, about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, or about 95%.

In some embodiments, altering the identity of a cysteine residue asdescribed in TABLE 1 can result in no greater than 1% aggregation, nogreater than 2% aggregation, no greater than 5% aggregation, no greaterthan 10% aggregation, no greater than 15% aggregation, no greater than20% aggregation, no greater than 25% aggregation, no greater than 30%aggregation, no greater than 35% aggregation, no greater than 40%aggregation, no greater than 45% aggregation, no greater than 50%aggregation, no greater than 55% aggregation, no greater than 60%aggregation, no greater than 65% aggregation, no greater than 70%aggregation, no greater than 75% aggregation, no greater than 80%aggregation, no greater than 85% aggregation, no greater than 90%aggregation, or no greater than 95% aggregation in solution.

In some cases, altering the identity of one or more amino acids canreduce the aggregation profile of a recombinant Arginase I in solution.In some cases, a recombinant Arginase I, or a functional fragmentthereof, comprises 1 amino acid mutation, 2 amino acid mutations, 3amino acid mutations, 4 amino acid mutations, 5 amino acid mutations, 6amino acid mutations, 7 amino acid mutations, 8 amino acid mutations, 9amino acid mutations, 10 amino acid mutations, 11 amino acid mutations,12 amino acid mutations, 13 amino acid mutations, 14 amino acidmutations, 15 amino acid mutations, 16 amino acid mutations, 17 aminoacid mutations, 18 amino acid mutations, 19 amino acid mutations, 20amino acid mutations, 21 amino acid mutations, 22 amino acid mutations,23 amino acid mutations, 24 amino acid mutations, 25 amino acidmutations, 26 amino acid mutations, 27 amino acid mutations, 28 aminoacid mutations, 29 amino acid mutations, 30 amino acid mutations, 31amino acid mutations, 32 amino acid mutations, 33 amino acid mutations,34 amino acid mutations, 35 amino acid mutations, 36 amino acidmutations, 37 amino acid mutations, 38 amino acid mutations, 39 aminoacid mutations, 40 amino acid mutations, 41 amino acid mutations, 42amino acid mutations, 43 amino acid mutations, 44 amino acid mutations,45 amino acid mutations, 46 amino acid mutations, 47 amino acidmutations, 48 amino acid mutations, 49 amino acid mutations, or 50 aminoacid mutations.

A recombinant Arginase, or a functional fragment thereof, can bepurified, for example, from bacterial cells, insect cells, mammaliancells, synthetic cells, or from cell-free systems. In some embodiments,the recombinant arginase is partially purified. In some embodiments, therecombinant arginase is substantially pure. In some embodiments, therecombinant arginase is at least 95% pure. In some embodiments, therecombinant arginase is 99% pure.

A purified recombinant arginase, or a functional fragment thereof, canbe at least 1% pure, at least 2% pure, at least 3% pure, at least 4%pure, at least 5% pure, at least 6% pure, at least 7% pure, at least 8%pure, at least 9% pure, at least 10% pure, at least 11% pure, at least12% pure, at least 13% pure, at least 14% pure, at least 15% pure, atleast 16% pure, at least 17% pure, at least 18% pure, at least 19% pure,at least 20% pure, at least 21% pure, at least 22% pure, at least 23%pure, at least 24% pure, at least 25% pure, at least 26% pure, at least27% pure, at least 28% pure, at least 29% pure, at least 30% pure, atleast 31% pure, at least 32% pure, at least 33% pure, at least 34% pure,at least 35% pure, at least 36% pure, at least 37% pure, at least 38%pure, at least 39% pure, at least 40% pure, at least 41% pure, at least42% pure, at least 43% pure, at least 44% pure, at least 45% pure, atleast 46% pure, at least 47% pure, at least 48% pure, at least 49% pure,at least 50% pure, at least 51% pure, at least 52% pure, at least 53%pure, at least 54% pure, at least 55% pure, at least 56% pure, at least57% pure, at least 58% pure, at least 59% pure, at least 60% pure, atleast 61% pure, at least 62% pure, at least 63% pure, at least 64% pure,at least 65% pure, at least 66% pure, at least 67% pure, at least 68%pure, at least 69% pure, at least 70% pure, at least 71% pure, at least72% pure, at least 73% pure, at least 74% pure, at least 75% pure, atleast 76% pure, at least 77% pure, at least 78% pure, at least 79% pure,at least 80% pure, at least 81% pure, at least 82% pure, at least 83%pure, at least 84% pure, at least 85% pure, at least 86% pure, at least87% pure, at least 88% pure, at least 89% pure, at least 90% pure, atleast 91% pure, at least 92% pure, at least 93% pure, at least 94% pure,at least 95% pure, at least 96% pure, at least 97% pure, at least 98%pure, at least 99% pure, at least 99.1% pure, at least 99.2% pure, atleast 99.3% pure, at least 99.4% pure, at least 99.5% pure, at least99.6% pure, at least 99.7% pure, at least 99.8% pure, or at least 99.9%pure.

Pegylated Recombinant Arginases.

An Arginase, or a functional fragment thereof, can be modified with oneor more polyethylene glycol molecule(s) (PEGs). The covalent attachmentof a PEG(s) oligomer to a drug or therapeutic protein can reduce theimmunogenicity and antigenicity of the recombinant Arginase I from thesubject's immune system. The covalent attachment of a PEG(s) oligomer toa drug or therapeutic protein can increase the hydrodynamic size of therecombinant Arginase, which can prolong the half-life of a pegylatedrecombinant human Arginase I in solution. PEG oligomers for use in thepresent invention can be, for example, —(CH₂CH₂O)_(n)— or—(CH₂CH₂O)_(n)—CH₂CH₂—, but can also include polyalkylene glycolsincluding, but not limited to polypropylene- or polybutylene glycols,methoxy poly(ethylene glycol), or methoxy poly(ethylene glycol)propionic acid (mPEG-acid) where n can be from about 1 to about 400.

An Arginase, or a functional fragment thereof, can be modified withvarious types of PEG molecules. In some embodiments, a PEG oligomer ismethoxy poly(ethylene glycol) succinimidyl proprionate (mPEG-SPA). Insome embodiments, a PEG oligomer is a methoxy poly(ethylene glycol)propionic acid (mPEG-acid). In some cases, the disclosure provides apharmaceutical composition comprising, a purified recombinant humanArginase I protein and at least one polyethylene glycol oligomer. Insome cases, the pegylated recombinant human Arginase I protein comprisesat least two polyethylene glycol oligomers. In some cases thepolyethylene glycol oligomer weighs from about 20 kilodaltons to about40 kilodaltons. In some cases the pegylated recombinant human Arginase Iprotein comprises from about 4 polyethylene glycol molecules to about 13polyethylene glycol oligomer. In some cases the polyethylene glycololigomer weighs about 5 kilodaltons.

The covalent attachment of an Arginase, or a functional fragmentthereof, to a polymer polyethylene glycol of interest can change thephysicochemical characteristics of the Arginase. Examples ofphysicochemical characteristics that can be altered by binding to a PEGinclude immunogenicity, in vitro and in vivo biological activity,absorption rate and bioavailability, biodistribuition, pharmacokinetic(PK) and pharmacodynamic profiles (PD), and toxicity. In someembodiments, a pegylated Arginase has a reduced immunogenicity. —NH₂,—COOH, —OH, —SH, and disulfide bonds are examples of chemical groups inthe amino acid side chain of an Arginase that could react with a PEGoligomer. The amine in the N-terminus and the carboxyl group in theC-terminus can also react with a PEG oligomer.

PEG reagents for protein pegylation can be activated PEGs. ActivatedPEGs can be used for amine pegylation, thiol pegylation, or N-terminalpegylation. PEG reagents are commercially available in differentlengths, shapes and chemistry allowing them to react with particularfunctional groups of proteins for their covalent attachment.Non-limiting examples of commercial suppliers of PEG include NOFCorporation (Japan); SunBio (South Korea); Chirotech Technology Limited(UK); JenKem (China); Creative PEGWorks (USA), Sigma-Aldrich (Milwaukee,Wis.), Dendritech (Midland, Mich.), or Polysciences™ (Warrington, Pa.).

Non-limiting examples of commercially available PEGs suitable for use inthe invention include, but are not limited to those available fromNektar Therapeutics, San Carlos, Calif., such as mPEG-NH₂ (Mw about 10kDa, about 20 kDa), methoxy PEG Succinimidyl α-Methylbutanoate (SMB),SMB-PEG-SMB, methoxy PEG Succinimidyl Propionate (mPEG-SPA), BranchedPEG N-Hydroxysuccinimide (mPEG2-NHS), mPEG-CM-HBA-NHS,NHS-HBA-CM-PEG-CM-HBA-NHS, mPEG-ButyrALD, ButyrALD-PEG-ButyrALD,Branched PEG ButyrALD (mPEG2-ButyrALD), Ortho-pyridylthioester(mPEG-OPTE), mPEG Maleimide (MAL), MAL-PEG-MAL, Branched PEG Maleimide(mPEG2-MAL), Forked Maleimide (mPEG-MAL2 and mPEG2-MAL2),mPEG-Ortho-pyridyldisulfide (mPEG-OPSS), OPSS-PEG-OPSS, mPEG-SH,SH-PEG-SH, Amine-PEG-Acid, Boc-PEG-NHS, Fmoc-PEG-NHS, MAL-PEG-NHS,Vinylsulfone-PEG-NHS, Acrylate-PEG-NHS Ester.

Non-limiting examples of PEGs that can be used in amine pegylationinclude, for example, PEGs manufactured by Jenken Technology USA suchas: Y-shape PEG NHS Esters, Y-shape PEG Carboxyl, Glucose PEG NHS Ester,Galactose PEG NHS Ester, Methoxy PEG Succinimidyl Carboxymethyl Ester,Methoxy PEG Carboxyl, Methoxy PEG Succinimidyl Butanoate, Methoxy PEGSuccinimidyl Hexanoate, Methoxy PEG Hexanoic Acid, Methoxy PEGSuccinimidyl Succinamide, Methoxy PEG Succinimidyl Glutaramide, MethoxyPEG Succinimidyl Carbonate, Methoxy PEG Nitrophenyl Carbonate, MethoxyPEG Succinimidyl Succinate, Methoxy PEG Succinimidyl Glutarate.Non-limiting examples of PEGs that can be used in thiol pegylationinclude Y-shape PEG Maleimide, Methoxy PEG Maleimide, Methoxy PEGVinylsulfone, Methoxy PEG Thiol. Non-limiting examples of PEGs that canbe used in N-terminal pegylation include, for example, PEGs manufacturedby Jenken Technology USA such as: Y-shape PEG Aldehyde, Y-shape PEGAcetaldehyde, Y-shape PEG Propionaldehyde, Methoxy PEG Propionaldehyde.

In some cases a recombinant Arginase, or a functional fragment thereof,can have a molecular weight that is small compared to the PEG oligomerto which it is attached. The molecular weight of a PEG oligomer can be,for example, no greater than 100 kilodaltons (kDa), no greater than 95kilodaltons, no greater than 90 kilodaltons, no greater 85 thankilodaltons (kDa), no greater than 80 kilodaltons (kDa), no greater than75 kilodaltons (kDa), no greater than 70 kilodaltons (kDa), no greaterthan 65 kilodaltons (kDa), no greater than 60 kilodaltons (kDa), nogreater than 55 kilodaltons (kDa), no greater than 50 kilodaltons (kDa),no greater than 45 kilodaltons (kDa), no greater than 40 kilodaltons(kDa), no greater than 35 kilodaltons (kDa), no greater than 30kilodaltons (kDa), no greater than 25 kilodaltons (kDa), no greater than20 kilodaltons (kDa), no greater than 15 kilodaltons (kDa), no greaterthan 10 kilodaltons (kDa), no greater than 5 kilodaltons (kDa), nogreater than 1 kilodalton (kDa), or no greater than 500 daltons (Da).

In some cases, the molecular weight of a PEG molecule can be greaterthan 500 daltons (Da), greater than 1 kilodalton (kDa), greater than 5kilodaltons (kDa), greater than 10 kilodaltons (kDa), greater than 15kilodaltons (kDa), greater than 20 kilodaltons (kDa), greater than 25kilodaltons (kDa), greater than 30 kilodaltons (kDa), greater than 35kilodaltons (kDa), greater than 40 kilodaltons (kDa), greater than 45kilodaltons (kDa), greater than 50 kilodaltons (kDa), greater than 55kilodaltons (kDa), greater than 60 kilodaltons (kDa), greater than 65kilodaltons (kDa), greater than 70 kilodaltons (kDa), greater than 75kilodaltons (kDa), greater than 80 kilodaltons (kDa), greater than 85kilodaltons (kDa), greater than 90 kilodaltons (kDa), greater than 95kilodaltons (kDa), greater than 100 kilodaltons (kDa).

In some cases the molecular weight of a PEG oligomer can be from about 1kilodalton (kDa) to about 5 kilodaltons (kDa), from about 1 kilodalton(kDa) to about 10 kilodaltons (kDa), from about 10 kilodaltons (kDa) toabout 20 kilodaltons (kDa), from about 10 kilodaltons (kDa) to about 30kilodaltons (kDa), from about 10 kilodaltons (kDa) to about 40kilodaltons (kDa), from about 10 kilodaltons (kDa) to about 50kilodaltons (kDa), from about 20 kilodaltons (kDa) to about 30kilodaltons (kDa), from about 20 kilodaltons (kDa) to about 40kilodaltons (kDa), from about 20 kilodaltons (kDa) to about 50kilodaltons (kDa), from about 30 kilodaltons (kDa) to about 40kilodaltons (kDa), from about 30 kilodaltons (kDa) to about 50kilodaltons (kDa).

In some embodiments, the molecular weight of a PEG oligomer is about 5kilodaltons (kDa). In some embodiments, the molecular weight of a PEGoligomer is from about 20 kilodaltons (kDa) to about 40 kilodaltons(kDa).

Recombinant Arginases Modified with PEG Oligomer(s).

The present disclosure provides PEG oligomers that can be attached torecombinant Arginases, such as SEQ ID NOs: 1-16 to modify or improverheology properties. PEG oligomers can also be attached to functionalfragments of the recombinant Arginases. The improvements can lead toimproved formulations. A PEG oligomer can be covalently attached to arecombinant Arginase. A PEG oligomer can be attached to the N-terminus,the C-terminus, or through a side-chain of the recombinant Arginase. Forexample, a PEG oligomer could be attached to a terminus of the aminoacid sequence of the recombinant Arginase, or could be attached to aside chain, such as the side chain of a lysine, serine, threonine,cysteine, tyrosine, aspartic acid, or glutamic acid residue. Theattachment can be via an amide bond, an ester bond, an ether bond, acarbamate bond, or a thioether bond.

In some cases a polyethylene glycol molecule(s) is conjugated to acysteine residue of a purified recombinant human Arginase I. In somecases a polyethylene glycol molecule is conjugated to an amine residueof a purified recombinant human Arginase I protein. In some cases apolyethylene glycol molecule is conjugated to the N-terminus of apurified recombinant human Arginase I protein. FIG. 14 illustrates aprocess that was utilized to evaluate the pegylation of a recombinanthuman Arginase I.

Pharmaceutical Compositions.

A pharmaceutical composition of the invention can be a combination ofany recombinant Arginase(s) described herein with other chemicalcomponents, such as carriers, stabilizers, diluents, dispersing agents,suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the recombinantArginase to an organism. Pharmaceutical compositions can be administeredin therapeutically-effective amounts as pharmaceutical compositions byvarious forms and routes including, for example, intravenous,subcutaneous, intramuscular, rectal, aerosol, parenteral, ophthalmic,pulmonary, transdermal, vaginal, optic, nasal, and topicaladministration. A pharmaceutical composition can be administered in alocal or systemic manner, for example, via injection of the recombinantArginase directly into an organ, optionally in a depot.

Parenteral injections can be formulated for bolus injection orcontinuous infusion. The pharmaceutical compositions can be in a formsuitable for parenteral injection as a sterile suspension, solution oremulsion in oily or aqueous vehicles, and can contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents. Pharmaceuticalformulations for parenteral administration include aqueous solutions ofthe recombinant Arginase(s) in water-soluble form. Suspensions of therecombinant Arginase(s) can be prepared as oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Aqueous injection suspensions can containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. The suspension canalso contain suitable stabilizers or agents which increase thesolubility and/or reduces the aggregation of the recombinant Arginase(s)to allow for the preparation of highly concentrated solutions.Alternatively, the recombinant Arginases can be lyophilized or in powderform for re-constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use. In some embodiments, a purifiedpegylated recombinant Arginase of the invention is administeredintravenously.

The recombinant Arginase(s) can be administered topically and can beformulated into a variety of topically administrable compositions, suchas solutions, suspensions, lotions, gels, pastes, medicated sticks,balms, creams, and ointments. Such pharmaceutical compositions cancontain solubilizers, stabilizers, tonicity enhancing agents, buffersand preservatives.

In practicing the methods of treatment or use provided herein,therapeutically-effective amounts of the recombinant Arginase(s)described herein are administered in pharmaceutical compositions to asubject suffering from a condition that affects the immune system. Insome embodiments, the subject is a mammal such as a human. Atherapeutically-effective amount can vary widely depending on theseverity of the disease, the age and relative health of the subject, thepotency of the compounds used, and other factors.

Pharmaceutical compositions can be formulated using one or morephysiologically-acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active compounds intopreparations that can be used pharmaceutically. Formulation can bemodified depending upon the route of administration chosen.Pharmaceutical compositions comprising a compounds described herein canbe manufactured, for example, by mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orcompression processes. The pharmaceutical compositions can include atleast one pharmaceutically acceptable carrier, diluent, or excipient andcompounds described herein as free-base or pharmaceutically-acceptablesalt form.

Methods for the preparation of recombinant Arginase(s) comprising thecompounds described herein include formulating the recombinantArginase(s) with one or more inert, pharmaceutically-acceptableexcipients or carriers to form a solid, semi-solid, or liquidcomposition. Solid compositions include, for example, powders, tablets,dispersible granules, capsules, cachets, and suppositories. Liquidcompositions include, for example, solutions in which a recombinantArginase(s) is dissolved, emulsions comprising a recombinantArginase(s), or a solution containing liposomes, micelles, ornanoparticles comprising a recombinant Arginase(s) as disclosed herein.Semi-solid compositions include, for example, gels, suspensions andcreams. The compositions can be in liquid solutions or suspensions,solid forms suitable for solution or suspension in a liquid prior touse, or as emulsions. These compositions can also contain minor amountsof nontoxic, auxiliary substances, such as wetting or emulsifyingagents, pH buffering agents, and other pharmaceutically-acceptableadditives.

Non-limiting examples of pharmaceutically-acceptable excipients can befound, for example, in Remington: The Science and Practice of Pharmacy,Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, JohnE., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999), each of which is incorporated by reference in itsentirety.

Methods of Administration.

Pharmaceutical compositions containing recombinant Arginase(s), orfunctional fragments of recombinant Arginases, described herein can beadministered for prophylactic and/or therapeutic treatments. Intherapeutic applications, the compositions can be administered to asubject already suffering from a disease or condition, in an amountsufficient to cure or at least partially arrest the symptoms of thedisease or condition, or to cure, heal, improve, or ameliorate thecondition. Recombinant Arginases(s) can also be administered to lessen alikelihood of developing, contracting, or worsening a condition. Amountseffective for this use can vary based on the severity and course of thedisease or condition, previous therapy, the subject's health status,weight, and response to the drugs, and the judgment of the treatingphysician. In some embodiments, the invention described herein providesa method of treating an inflammatory disease in a subject, the methodcomprising administering to the subject a therapeutically-effectiveamount of a purified recombinant arginase. In some embodiments, theinflammatory disease is rheumatoid arthritis. In some embodiments, theinflammatory disease is multiple sclerosis. In some embodiments, theinflammatory disease is a chronic or acute inflammation in a bone. Insome embodiments, the purified recombinant arginase is a pegylatedrecombinant human Arginase I. In some embodiments, the human Arginase Iis SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, or SEQ ID NO: 16. In some embodiments, the pegylated recombinanthuman Arginase I comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

Multiple recombinant Arginase(s) can be administered in any order orsimultaneously. In some cases, multiple functional fragments ofrecombinant Arginases can be administered in any order orsimultaneously. If simultaneously, the multiple recombinant Arginase(s)can be provided in a single, unified form, such as an intravenousinjection, or in multiple forms, for example, as multiple intravenousinjections or pills. The recombinant Arginase(s) can be packed togetheror separately, in a single package or in a plurality of packages. One orall of the recombinant Arginase(s) can be given in multiple doses. Ifnot simultaneous, the timing between the multiple doses may vary to asmuch as about a month.

Compounds and compositions of the invention can be packaged as a kit. Insome embodiments, a kit includes written instructions on the use of thecompounds and compositions. In some embodiments the invention provides amethod of modulating inflammation, the method comprising administeringto a subject a therapeutically-effective amount of a purified pegylatedrecombinant human Arginase I, wherein the administration modulates theinflammation. In some embodiments the therapeutically-effective amountof a purified pegylated recombinant human Arginase I is administered forat least 24 hours. In some embodiments the therapeutically-effectiveamount of a purified pegylated recombinant human Arginase I isadministered for at least one week. In some embodiments thetherapeutically-effective amount of a purified pegylated recombinanthuman Arginase I is administered for at least two weeks.

Recombinant Arginase(s), or functional fragments thereof, describedherein can be administered before, during, or after the occurrence of adisease or condition, and the timing of administering the compositioncontaining a recombinant Arginase(s) can vary. For example, therecombinant Arginase(s) can be used as a prophylactic and can beadministered continuously to subjects with a propensity to conditions ordiseases in order to lessen a likelihood of the occurrence of thedisease or condition. The recombinant Arginase(s) can be administered toa subject during or as soon as possible after the onset of the symptoms.The administration of the recombinant Arginases(s) can be initiatedimmediately within the onset of symptoms, within the first 3 hours ofthe onset of the symptoms, within the first 6 hours of the onset of thesymptoms, within the first 24 hours of the onset of the symptoms, within48 hours of the onset of the symptoms, or within any period of time fromthe onset of symptoms. The initial administration can be via any routepractical, such as by any route described herein using any formulationdescribed herein. In some embodiments, the administration of a pegylatedrecombinant human Arginase I of the disclosure is an intravenousadministration. A recombinant Arginase(s) can be administered as soon asis practicable after the onset of an immune disease or condition isdetected or suspected, and for a length of time necessary for thetreatment of the immune disease, such as, for example, from about 24hours to about 48 hours, from about 48 hours to about 1 week, from about1 week to about 2 weeks, from about 2 weeks to about 1 month, from about1 month to about 3 months. In some embodiments, a recombinantArginase(s) can be administered for at least 24 hours, at least 48hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2months, at least 3 months, at least 4 months, at least 5 months, atleast 6 months, at least 7 months, at least 8 months, at least 9 months,at least 10 months, at least 11 months, at least 12 months, at least 1year, at least 2 years at least 3 years, at least 4 years, or at least 5years. The length of treatment can vary for each subject. In someembodiments, pegylation of the recombinant Arginase modulates thehalf-life of the recombinant Arginase in vivo.

Dosages.

Pharmaceutical compositions described herein can be in unit dosage formssuitable for single administration of precise dosages. In unit dosageform, the formulation is divided into unit doses containing appropriatequantities of one or more compounds. The unit dosage can be in the formof a package containing discrete quantities of the formulation.Non-limiting examples are packaged tablets or capsules, and powders invials or ampoules. Aqueous suspension compositions can be packaged insingle-dose non-reclosable containers. Multiple-dose reclosablecontainers can be used, for example, in combination with a preservativeor without a preservative. In some embodiments, the pharmaceuticalcomposition does not comprise a preservative. Formulations forparenteral injection can be presented in unit dosage form, for example,in ampoules, or in multi-dose containers with a preservative.

A recombinant Arginase(s), or a functional fragment thereof, describedherein can be present in a composition in a range of from about 1 mg toabout 2000 mg; from about 5 mg to about 1000 mg, from about 10 mg toabout 500 mg, from about 50 mg to about 250 mg, from about 100 mg toabout 200 mg, from about 1 mg to about 50 mg, from about 50 mg to about100 mg, from about 100 mg to about 150 mg, from about 150 mg to about200 mg, from about 200 mg to about 250 mg, from about 250 mg to about300 mg, from about 300 mg to about 350 mg, from about 350 mg to about400 mg, from about 400 mg to about 450 mg, from about 450 mg to about500 mg, from about 500 mg to about 550 mg, from about 550 mg to about600 mg, from about 600 mg to about 650 mg, from about 650 mg to about700 mg, from about 700 mg to about 750 mg, from about 750 mg to about800 mg, from about 800 mg to about 850 mg, from about 850 mg to about900 mg, from about 900 mg to about 950 mg, or from about 950 mg to about1000 mg.

A recombinant Arginase(s), or a functional fragment thereof, describedherein can be present in a composition in an amount of about 1 mg, about2 mg, about 3 mg, about 4 mg, about 5 mg, about 10 mg, about 15 mg,about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg,about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about1000 mg, about 1050 mg, about 1100 mg, about 1150 mg, about 1200 mg,about 1250 mg, about 1300 mg, about 1350 mg, about 1400 mg, about 1450mg, about 1500 mg, about 1550 mg, about 1600 mg, about 1650 mg, about1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900 mg,about 1950 mg, or about 2000 mg.

The therapeutically effective dose of a pegylated recombinant Arginase,or a functional fragment thereof, of the invention can be from about 1ng/kg to about 10 ng/kg, from about 1 ng/kg to about 100 ng/kg, fromabout 1 ng/kg to about 1 mg/kg, from about 1 ng/kg to about 10 mg/kg,from about 1 ng/kg to about 100 mg/kg, from about 1 ng/kg to about 250mg/kg, from about 1 ng/kg to about 500 mg/kg, from about 1 ng/kg toabout 750 mg/kg, from about 1 ng/kg to about 1,000 mg/kg, from about 1ng/kg to about 1,250 mg/kg, from about 1 ng/kg to about 1,500 mg/kg,from about 1 ng/kg to about 1,750 mg/kg, from about 1 ng/kg to about2,000 mg/kg, from about 10 ng/kg to about 100 ng/kg, from about 10 ng/kgto about 1 mg/kg, from about 10 ng/kg to about 10 mg/kg, from about 10ng/kg to about 100 mg/kg, from about 10 ng/kg to about 500 mg/kg, fromabout 10 ng/kg to about 750 mg/kg, from about 10 ng/kg to about 1,000mg/kg, from about 10 ng/kg to about 1,250 mg/kg, from about 10 ng/kg toabout 1,500 mg/kg, from about 10 ng/kg to about 2,000 mg/kg, from about100 ng/kg to about 1 mg/kg, from about 100 ng/kg to about 10 mg/kg, fromabout 100 ng/kg to about 100 mg/kg, from about 100 ng/kg to about 250mg/kg, from about 100 ng/kg to about 500 mg/kg, from about 100 ng/kg toabout 750 mg/kg, from about 100 ng/kg to about 1,000 mg/kg, from about100 ng/kg to about 1,250 mg/kg, from about 100 ng/kg to about 1,500mg/kg, from about 100 ng/kg to about 1,750 mg/kg, from about 100 ng/kgto about 2,000 mg/kg, from about 1 mg/kg to about 10 mg/kg, from about 1mg/kg to about 100 mg/kg, from about 1 mg/kg to about 500 mg/kg, fromabout 1 mg/kg to about 750 mg/kg, from about 1 mg/kg to about 1,000mg/kg, from about 1 mg/kg to about 1,250 mg/kg, from about 1 mg/kg toabout 1,500 mg/kg, from about 1 mg/kg to about 1,750 mg/kg, from about 1mg/kg to about 2,000 mg/kg, from about 10 mg/kg to about 100 mg/kg, fromabout 10 mg/kg to about 500 mg/kg, from about 10 mg/kg to about 750mg/kg, from about 10 mg/kg to about 1,000 mg/kg, from about 10 mg/kg toabout 1,250 mg/kg, from about 10 mg/kg to about 1,500 mg/kg, from about10 mg/kg to about 1,750 mg/kg, from about 10 mg/kg to about 2,000 mg/kg,from about 100 mg/kg to about 500 mg/kg, from about 100 mg/kg to about750 mg/kg, from about 100 mg/kg to about 1,000 mg/kg, from about 100mg/kg to about 1,250 mg/kg, from about 100 mg/kg to about 1,500 mg/kg,from about 100 mg/kg to about 1,750 mg/kg, from about 100 mg/kg to about2,000 mg/kg, from about 500 mg/kg to about 750 mg/kg, from about 500mg/kg to about 1,000 mg/kg, from about 500 mg/kg to about 1,250 mg/kg,from about 500 mg/kg to about 1,500 mg/kg, from about 500 mg/kg to about1,750 mg/kg, from about 500 mg/kg to about 2,000 mg/kg, from about 750mg/kg to about 1,000 mg/kg, from about 750 mg/kg to about 1,250 mg/kg,from about 750 mg/kg to about 1,500 mg/kg, from about 750 mg/kg to about1,750 mg/kg, from about 750 mg/kg to about 2,000 mg/kg, from about 1,000mg/kg to about 1,250 mg/kg, from about 1,000 mg/kg to about 1,500 mg/kg,from about 1,000 mg/kg to about 1,750 mg/kg, or from about 1,000 mg/kgto about 2,000 mg/kg.

In some embodiments, the therapeutically-effective amount of a purifiedpegylated recombinant human Arginase I, or a functional fragmentthereof, is from about 1 mg/kg to about 10 mg/kg. In some embodiments,the therapeutically-effective amount of the purified pegylatedrecombinant human Arginase I is from about 10 mg/kg to about 100 mg/kg.In some embodiments, the therapeutically-effective amount of thepurified pegylated recombinant human Arginase I, or a functionalfragment thereof, is greater than 100 mg/kg.

Pharmacokinetic and Pharmacodynamic Measurements.

Pharmacokinetic and pharmacodynamic data can be obtained by variousexperimental techniques. Appropriate pharmacokinetic and pharmacodynamicprofile components describing a particular composition can vary due tovariations in drug metabolism in different subjects. Pharmacokinetic andpharmacodynamic profiles can be based on the determination of the meanparameters of a group of subjects. The group of subjects includes anyreasonable number of subjects suitable for determining a representativemean, for example, 5 subjects, 10 subjects, 15 subjects, 20 subjects, 25subjects, 30 subjects, 35 subjects, or more. The mean is determined bycalculating the average of all subject's measurements for each parametermeasured.

A dose can be modulated to achieve a desired pharmacokinetic orpharmacodynamics profile, such as a desired or effective blood profile,as described herein. To better characterize the enzyme kinetics ofrecombinant human Arginase I in vitro the K_(m), V_(max), K_(cat), andK_(cat)/K_(m) of five different recombinant Arginases were measured. Thesummary of the enzyme kinetics study for five human recombinantArginases is shown in TABLE 2.

TABLE 2 K_(m) V_(max) K_(cat) K_(cat)/K_(m) Mutant (mM) (μmol * ml⁻¹ *min⁻¹) (sec⁻¹) (mM⁻¹sec⁻¹) SEQ ID NO: 1 2.37 0.037 546.4 229.8 SEQ IDNO: 5 1.80 0.027 397 220.4 SEQ ID NO: 6 2.19 0.034 498 226.7 SEQ ID NO:8 2.59 0.038 562.8 216.8 SEQ ID NO: 7 2.02 0.032 470.8 232.9

The pharmacokinetics parameters can be any parameters suitable fordescribing the plasma profiles of a recombinant Arginase I, or afunctional fragment thereof, of the invention. For example, thepharmacokinetics profile can be obtained at a time after dosing of, forexample, about zero minutes, about 1 minute, about 2 minutes, about 3minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39minutes, about 40 minutes, about 41 minutes, about 42 minutes, about 43minutes, about 44 minutes, about 45 minutes, about 46 minutes, about 47minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59minutes, about 60 minutes, about zero hours, about 0.5 hours, about 1hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours,about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours,about 10 hours, about 10.5 hours, about 11 hours, about 11.5 hours,about 12 hours, about 12.5 hours, about 13 hours, about 13.5 hours,about 14 hours, about 14.5 hours, about 15 hours, about 15.5 hours,about 16 hours, about 16.5 hours, about 17 hours, about 17.5 hours,about 18 hours, about 18.5 hours, about 19 hours, about 19.5 hours,about 20 hours, about 20.5 hours, about 21 hours, about 21.5 hours,about 22 hours, about 22.5 hours, about 23 hours, about 23.5 hours, orabout 24 hours.

The pharmacokinetic parameters can be any parameters suitable fordescribing a recombinant Arginase I, or a functional fragment thereof.The C_(max) can be, for example, not less than about 1 μg/mL; not lessthan about 5 μg/mL; not less than about 10 μg/mL; not less than about 15μg/mL; not less than about 20 μg/mL; not less than about 25 μg/mL; notless than about 50 μg/mL; not less than about 75 μg/mL; not less thanabout 100 μg/mL; not less than about 200 μg/mL; not less than about 300μg/mL; not less than about 400 μg/mL; not less than about 500 μg/mL; notless than about 600 μg/mL; not less than about 700 μg/mL; not less thanabout 800 μg/mL; not less than about 900 μg/mL; not less than about 1000μg/mL; not less than about 1250 μg/mL; not less than about 1500 μg/mL;not less than about 1750 μg/mL; not less than about 2000 μg/mL; or anyother C_(max) appropriate for describing a pharmacokinetic profile of anArginase described herein. The C_(max) can be, for example, about 1μg/mL to about 5,000 μg/mL; about 1 μg/mL to about 4,500 μg/mL; about 1μg/mL to about 4,000 μg/mL; about 1 μg/mL to about 3,500 μg/mL; about 1μg/mL to about 3,000 μg/mL; about 1 μg/mL to about 2,500 μg/mL; about 1μg/mL to about 2,000 μg/mL; about 1 μg/mL to about 1,500 μg/mL; about 1μg/mL to about 1,000 μg/mL; about 1 μg/mL to about 900 μg/mL; about 1μg/mL to about 800 μg/mL; about 1 μg/mL to about 700 μg/mL; about 1μg/mL to about 600 μg/mL; about 1 μg/mL to about 500 μg/mL; about 1μg/mL to about 450 μg/mL; about 1 μg/mL to about 400 μg/mL; about 1μg/mL to about 350 μg/mL; about 1 μg/mL to about 300 μg/mL; about 1μg/mL to about 250 μg/mL; about 1 μg/mL to about 200 μg/mL; about 1μg/mL to about 150 μg/mL; about 1 μg/mL to about 125 μg/mL; about 1μg/mL to about 100 μg/mL; about 1 μg/mL to about 90 μg/mL; about 1 μg/mLto about 80 μg/mL; about 1 μg/mL to about 70 μg/mL; about 1 μg/mL toabout 60 μg/mL; about 1 μg/mL to about 50 μg/mL; about 1 μg/mL to about40 μg/mL; about 1 μg/mL to about 30 μg/mL; about 1 μg/mL to about 20μg/mL; about 1 μg/mL to about 10 μg/mL; about 1 μg/mL to about 5 μg/mL;about 10 μg/mL to about 4,000 μg/mL; about 10 μg/mL to about 3,000μg/mL; about 10 μg/mL to about 2,000 μg/mL; about 10 μg/mL to about1,500 μg/mL; about 10 μg/mL to about 1,000 μg/mL; about 10 μg/mL toabout 900 μg/mL; about 10 μg/mL to about 800 μg/mL; about 10 μg/mL toabout 700 μg/mL; about 10 μg/mL to about 600 μg/mL; about 10 μg/mL toabout 500 μg/mL; about 10 μg/mL to about 400 μg/mL; about 10 μg/mL toabout 300 μg/mL; about 10 μg/mL to about 200 μg/mL; about 10 μg/mL toabout 100 μg/mL; about 10 μg/mL to about 50 μg/mL; about 25 μg/mL toabout 500 μg/mL; about 25 μg/mL to about 100 μg/mL; about 50 μg/mL toabout 500 μg/mL; about 50 μg/mL to about 100 μg/mL; about 100 μg/mL toabout 500 μg/mL; about 100 μg/mL to about 400 μg/mL; about 100 μg/mL toabout 300 μg/mL; or about 100 μg/mL to about 200 μg/mL.

The T_(max) of an Arginase I, or a functional fragment thereof,described herein can be, for example, not greater than about 0.5 hours,not greater than about 1 hours, not greater than about 1.5 hours, notgreater than about 2 hours, not greater than about 2.5 hours, notgreater than about 3 hours, not greater than about 3.5 hours, notgreater than about 4 hours, not greater than about 4.5 hours, notgreater than about 5 hours, or any other T_(max) appropriate fordescribing a pharmacokinetic profile of a compound described herein. TheT_(max) can be, for example, about 0.1 hours to about 24 hours; about0.1 hours to about 0.5 hours; about 0.5 hours to about 1 hour; about 1hour to about 1.5 hours; about 1.5 hours to about 2 hour; about 2 hoursto about 2.5 hours; about 2.5 hours to about 3 hours; about 3 hours toabout 3.5 hours; about 3.5 hours to about 4 hours; about 4 hours toabout 4.5 hours; about 4.5 hours to about 5 hours; about 5 hours toabout 5.5 hours; about 5.5 hours to about 6 hours; about 6 hours toabout 6.5 hours; about 6.5 hours to about 7 hours; about 7 hours toabout 7.5 hours; about 7.5 hours to about 8 hours; about 8 hours toabout 8.5 hours; about 8.5 hours to about 9 hours; about 9 hours toabout 9.5 hours; about 9.5 hours to about 10 hours; about 10 hours toabout 10.5 hours; about 10.5 hours to about 11 hours; about 11 hours toabout 11.5 hours; about 11.5 hours to about 12 hours; about 12 hours toabout 12.5 hours; about 12.5 hours to about 13 hours; about 13 hours toabout 13.5 hours; about 13.5 hours to about 14 hours; about 14 hours toabout 14.5 hours; about 14.5 hours to about 15 hours; about 15 hours toabout 15.5 hours; about 15.5 hours to about 16 hours; about 16 hours toabout 16.5 hours; about 16.5 hours to about 17 hours; about 17 hours toabout 17.5 hours; about 17.5 hours to about 18 hours; about 18 hours toabout 18.5 hours; about 18.5 hours to about 19 hours; about 19 hours toabout 19.5 hours; about 19.5 hours to about 20 hours; about 20 hours toabout 20.5 hours; about 20.5 hours to about 21 hours; about 21 hours toabout 21.5 hours; about 21.5 hours to about 22 hours; about 22 hours toabout 22.5 hours; about 22.5 hours to about 23 hours; about 23 hours toabout 23.5 hours; or about 23.5 hours to about 24 hours.

The AUC_((0-inf)) of an Arginase I, or a functional fragment thereof,described herein can be, for example, not less than about 100 μg·hr/mL,not less than about 125 μg·hr/mL, not less than about 150 μg·hr/mL, notless than about 175 μg·hr/mL, not less than about 200 μg·hr/mL, not lessthan about 250 μg·hr/mL, not less than about 300 μg·hr/mL, not less thanabout 350 μg·hr/mL, not less than about 400 μg·hr/mL, not less thanabout 500 μg·hr/mL, not less than about 600 μg·hr/mL, not less thanabout 700 μg·hr/mL, not less than about 800 μg·hr/mL, not less thanabout 900 μg·hr/mL, not less than about 1000 μg·hr/mL, not less thanabout 2000 μg·hr/mL, not less than about 3000 μg·hr/mL, not less thanabout 4000 μg·hr/mL, not less than about 5000 μg·hr/mL, not less thanabout 6000 μg·hr/mL, not less than about 7000 μg·hr/mL, not less thanabout 8000 μg·hr/mL, not less than about 9000 μg·hr/mL, not less thanabout 10000 μg·hr/mL, not less than about 11000 μg·hr/mL, not less thanabout 12000 μg·hr/mL, not less than about 13000 μg·hr/mL, not less thanabout 14000 μg·hr/mL, not less than about 15000 μg·hr/mL, not less thanabout 16000 μg·hr/mL, not less than about 17000 μg·hr/mL, not less thanabout 18000 μg·hr/mL, not less than about 19000 μg·hr/mL, not less thanabout 20000 μg·hr/mL, not less than about 21000 μg·hr/mL, not less thanabout 22000 μg·hr/mL, not less than about 23000 μg·hr/mL, not less thanabout 24000 μg·hr/mL, not less than about 25000 μg·hr/mL, not less thanabout 26000 μg·hr/mL, not less than about 27000 μg·hr/mL, not less thanabout 28000 μg·hr/mL, not less than about 29000 μg·hr/mL, not less thanabout 30000 μg·hr/mL, not less than about 31000 μg·hr/mL, not less thanabout 32000 μg·hr/mL, not less than about 33000 μg·hr/mL, not less thanabout 34000 μg·hr/mL, not less than about 35000 μg·hr/mL, or any otherAUC_((0-inf)) appropriate for describing a pharmacokinetic profile of anArginase described herein.

The AUC_((0-inf)) of an Arginase I, or a functional fragment thereof,described herein can be, for example, about 1,000 μg·hr/mL to about1,250 μg·hr/mL; about 1,250 μg·hr/mL to about 1,500 μg·hr/mL; about1,500 μg·hr/mL to about 1,750 μg·hr/mL; about 1,750 μg·hr/mL to about2,000 μg·hr/mL; about 2,000 μg·hr/mL to about 2,500 μg·hr/mL; about2,500 μg·hr/mL to about 3,000 μg·hr/mL; about 3,000 μg·hr/mL to about3,500 μg·hr/mL; about 3,500 μg·hr/mL to about 4,000 μg·hr/mL; about4,000 μg·hr/mL to about 4,500 μg·hr/mL; about 4,500 μg·hr/mL to about5,000 μg·hr/mL; about 5,000 μg·hr/mL to about 5,500 μg·hr/mL; about5,500 μg·hr/mL to about 6,000 μg·hr/mL; about 6,000 μg·hr/mL to about6,500 μg·hr/mL; about 6,500 μg·hr/mL to about 7,000 μg·hr/mL; about7,000 μg·hr/mL to about 7,500 μg·hr/mL; about 7,500 μg·hr/mL to about8,000 μg·hr/mL; about 8,000 μg·hr/mL to about 8,500 μg·hr/mL; about8,500 μg·hr/mL to about 9,000 μg·hr/mL; about 9,000 μg·hr/mL to about9,500 μg·hr/mL; about 9,500 μg·hr/mL to about 10,000 μg·hr/mL; about10,000 μg·hr/mL to about 20,000 μg·hr/mL; about 20,000 μg·hr/mL to about30,000 μg·hr/mL; about 30,000 μg·hr/mL to about 40,000 μg·hr/mL; about40,000 μg·hr/mL to about 50,000 μg·hr/mL; about 50,000 μg·hr/mL to about60,000 μg·hr/mL; about 60,000 μg·hr/mL to about 70,000 μg·hr/mL; about70,000 μg·hr/mL to about 80,000 μg·hr/mL; about 80,000 μg·hr/mL to about90,000 μg·hr/mL; or about 90,000 μg·hr/mL to about 100,000 μg·hr/mL.

The plasma concentration of a recombinant human Arginase I, or afunctional fragment thereof, described herein can be, for example, notless than about 1 g/mL, not less than about 2 μg/mL, not less than about3 μg/mL, not less than about 4 μg/mL, not less than about 5 μg/mL, notless than about 6 μg/mL, not less than about 7 μg/mL, not less thanabout 8 μg/mL, not less than about 9 μg/mL, not less than about 10μg/mL, not less than about 11 μg/mL, not less than about 12 μg/mL, notless than about 13 μg/mL, not less than about 14 μg/mL, not less thanabout 15 μg/mL, not less than about 16 μg/mL, not less than about 17μg/mL, not less than about 18 μg/mL, not less than about 19 μg/mL, notless than about 20 μg/mL, not less than about 21 μg/mL, not less thanabout 22 μg/mL, not less than about 23 μg/mL, not less than about 24μg/mL, not less than about 25 μg/mL, not less than about 26 μg/mL, notless than about 27 μg/mL, not less than about 28 μg/mL, not less thanabout 29 μg/mL, not less than about 30 μg/mL, not less than about 31μg/mL, not less than about 32 μg/mL, not less than about 33 μg/mL, notless than about 34 μg/mL, not less than about 35 μg/mL, not less thanabout 36 μg/mL, not less than about 37 μg/mL, not less than about 38μg/mL, not less than about 39 μg/mL, not less than about 40 μg/mL, notless than about 41 μg/mL, not less than about 42 μg/mL, not less thanabout 43 μg/mL, not less than about 44 μg/mL, not less than about 45μg/mL, not less than about 46 μg/mL, not less than about 47 μg/mL, notless than about 48 μg/mL, not less than about 49 μg/mL, not less thanabout 50 μg/mL, not less than about 51 μg/mL, not less than about 52μg/mL, not less than about 53 μg/mL, not less than about 54 μg/mL, notless than about 55 μg/mL, not less than about 56 μg/mL, not less thanabout 57 μg/mL, not less than about 58 μg/mL, not less than about 59μg/mL, not less than about 60 μg/mL, not less than about 61 μg/mL, notless than about 62 μg/mL, not less than about 63 μg/mL, not less thanabout 64 μg/mL, not less than about 65 μg/mL, not less than about 66μg/mL, not less than about 67 μg/mL, not less than about 68 μg/mL, notless than about 69 μg/mL, not less than about 70 μg/mL, not less thanabout 71 μg/mL, not less than about 72 μg/mL, not less than about 73μg/mL, not less than about 74 μg/mL, not less than about 75 μg/mL, notless than about 76 μg/mL, not less than about 77 μg/mL, not less thanabout 78 μg/mL, not less than about 79 μg/mL, not less than about 80μg/mL, not less than about 81 μg/mL, not less than about 82 μg/mL, notless than about 83 μg/mL, not less than about 84 μg/mL, not less thanabout 85 μg/mL, not less than about 86 μg/mL, not less than about 87μg/mL, not less than about 88 μg/mL, not less than about 89 μg/mL, notless than about 90 μg/mL, not less than about 91 μg/mL, not less thanabout 92 μg/mL, not less than about 93 μg/mL, not less than about 94μg/mL, not less than about 95 μg/mL, not less than about 96 μg/mL, notless than about 97 μg/mL, not less than about 98 μg/mL, not less thanabout 99 μg/mL, not less than about 100 μg/mL, not less than about 105μg/mL, not less than about 110 μg/mL, not less than about 115 μg/mL, notless than about 120 μg/mL, not less than about 125 μg/mL, not less thanabout 130 μg/mL, not less than about 135 μg/mL, not less than about 140μg/mL, not less than about 145 μg/mL, not less than about 150 μg/mL, notless than about 155 μg/mL, not less than about 160 μg/mL, not less thanabout 165 μg/mL, not less than about 170 μg/mL, not less than about 175μg/mL, not less than about 180 μg/mL, not less than about 185 μg/mL, notless than about 190 μg/mL, not less than about 195 μg/mL, not less thanabout 200 μg/mL, not less than about 205 μg/mL, not less than about 210μg/mL, not less than about 215 μg/mL, not less than about 220 μg/mL, notless than about 225 μg/mL, not less than about 230 μg/mL, not less thanabout 235 μg/mL, not less than about 240 μg/mL, not less than about 245μg/mL, not less than about 250 μg/mL, or any other plasma concentrationof a compound described herein.

The plasma concentration can be, for example, about 1 μg/mL to about 2μg/mL; about 1 μg/mL to about 5 μg/mL; about 5 μg/mL to about 10 μg/mL;about 10 μg/mL to about 25 μg/mL; about 25 μg/mL to about 50 μg/mL;about 50 μg/mL to about 75 μg/mL; about 75 ng/mL to about 100 μg/mL;about 100 μg/mL to about 150 μg/mL; about 100 μg/mL to about 200 μg/mLabout 150 μg/mL to about 200 μg/mL; about 200 μg/mL to about 250 μg/mL;about 250 μg/mL to about 300 μg/mL; about 300 μg/mL to about 350 μg/mL;about 350 μg/mL to about 400 μg/mL; about 400 μg/mL to about 450 μg/mL;about 450 μg/mL to about 500 μg/mL; about 500 μg/mL to about 600 μg/mL;about 600 μg/mL to about 700 μg/mL; about 700 μg/mL to about 800 μg/mL;about 800 μg/mL to about 900 μg/mL; about 900 μg/mL to about 1,000μg/mL; about 1,000 μg/mL to about 1,100 μg/mL; about 1,100 μg/mL toabout 1,200 μg/mL; about 1,200 μg/mL to about 1,300 μg/mL; about 1,300μg/mL to about 1,400 μg/mL; about 1,400 μg/mL to about 1,500 μg/mL;about 1,500 μg/mL to about 1,600 μg/mL; about 1,600 μg/mL to about 1,700μg/mL; about 1,700 μg/mL to about 1,800 μg/mL; about 1,800 μg/mL toabout 1,900 μg/mL; or about 1,900 μg/mL to about 2,000 μg/mL.

Example 1. Modulating the Immune System with Recombinant Arginase I

The following experiments were conducted to characterize the function ofa purified Arginase in the modulation of an immune condition, such as acondition associated with an inflammation.

Materials and Methods.

Mice: floxed PTEN mice are described by Tak W. Mak (Suzuki, A. et al.2001. T cell-specific loss of Pten leads to defects in central andperipheral tolerance. Immunity 14: 523-534). LysM cre mice wereoriginally described by Clausen et al. (Clausen, B. E. et al. TransgenicRes. 8: 265-277). Littermate-controlled experiments were performed with8-12-week-old wildtype, floxed PTEN cre positive and cre negative miceon a C57Bl/6 background, which were backcrossed for at least 10generations. DBA mice were used in the collagen induced arthritisexperiments. All animals were bred and housed in a SPF facility of theMedical University of Vienna with a 12 h/12 h day/night cycle andconstant temperature. Mice of both sexes were used and nogender-specific differences were found.

Genotyping: mice were earmarked 3-4 weeks after birth. DNA from lysed(proteinase K lysis buffer) ear tissue was subjected to direct PCR usingGoTaq Polymerase (Promega™). Specific PCRs were performed with followingprimers: PTEN primer: forward, 5′-CTCCTCTACTCCATTCTTCCC-3′ (SEQ ID NO:17), reverse, 5′-ACTCCCACCAATGAACAAAC-3′ (SEQ ID NO: 18); cre primer:forward, 5′-TCGCGATTATCTTCTATATCTTCAG-3′ (SEQ ID NO: 19), reverse,5′-GCTCGACCAGTTTAGTTACCC-3′ (SEQ ID NO: 20).

Preparation and cultivation of primary macrophages:thioglycollate-elicited peritoneal macrophages were generated byinjection of 2 ml 4% thioglycollate (Sigma™) into the peritoneal cavityfollowed by peritoneal lavage with empty medium 3 days later. Isolatedmacrophages were seeded at a concentration of 1×10⁶ cells/ml inRPMI-1640 medium (Invitrogen™) supplemented with 10% FCS, 1%penicillin/streptomycin/fungizone, 1% L-glutamine, and cultured at 37°C. in a 5% CO₂ atmosphere. Cells were allowed to recover overnight andin-vitro stimulations were carried out by supplementing the macrophageculture with following agents and cytokines: 100 ng/ml ultrapure E. coliO111:B4 lipopolysaccharide (LPS) (Invivogen™), 100 nM wortmannin(Sigma™), 10 or 200 μM N-hydroxy-L-arginine (Calbiochem™) or 5 ng/mlrecombinant mouse IL-4/IL-13 (R&D Systems™). Stimulations were carriedout for 3, 8 or 24 hrs for RNA extraction, ABCD assay or proteinisolation and cytokine measurement, respectively.

Preparation and cultivation of bone marrow-derived dendritic cells(BMDC): bone marrow cells were flushed from femurs and tibias ofindicated mice and cultivated in complete RPMI medium supplemented with20 ng/ml recombinant mouse GM-CSF (R&D Systems™) at 37° C. in a 5% CO₂atmosphere. Half of the medium was replaced with fresh mediumsupplemented with 20 ng/ml GM-CSF at day 3 and day 6 after isolation.Dendritic cells (DC) were harvested and activated at day 7 and thematuration status was determined by flow cytometry using fluorescentconjugated antibodies against CD80 (PE-Cy5™) and MHC-II (PerCP-eFluor710™) (eBioscience™). Samples were incubated with the antibody-mix for15 min at 4° C. and subsequently acquired on a LSRII Flow Cytometer(Beckton Dickinson™). Data were analyzed using FlowJo™ Software 10.0(Treestar™) software.

Immunoblotting: cells were homogenized and lysed in Laemmli buffer.Proteins were separated by SDS-PAGE on a 10% denaturing polyacrylamidegel, which was stained with Coomassie Brilliant Blue (Thermo ScientificPierce™) after electrophoresis. Proteins were blotted onto apolyvinylidene difluoride membrane (PVDF™, Millipore™) and, afterblocking with 5% dry milk/0.1% Tween 20, incubated overnight withprimary antibody. Following antibodies were used: chicken anti-Arginase1 (kindly provided by Dr. Morris), rabbit antibodies against iNOS(NEB™), PTEN, STAT6, pSTAT6 (Cell Signaling Technology™), C/EBPβ (SantaCruz Biotech) or β-Actin (Sigma™). After incubation for 2 hrs at roomtemperature (RT) with the respective peroxidase-conjugated secondaryantibody and following development with SuperSignal West Femto(Pierce™), signals were detected using chemiluminescence (FluorChem HD2chemiluminescence imager, Alpha Innotech™). Bands were analyzedaccording to their molecular weight.

Avidin-biotin complex DNA (ABCD) assay: cells were lysed with lysisbuffer (10 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA pH 8.0, 10% Glycerol,0.5% NP-40, 1 mM DTT, protease-inhibitor), sonicated (6 impulses for 2seconds), centrifuged and supernatants were incubated with buffer H (20mM HEPES pH 7.9, 50 mM KCl, 20% Glycerol, 1 mM DTT, 0.1% NP-40), 2. μg5′-biotinylated oligo and 20. μg herring sperm DNA for 5 min at 37° C.followed by incubation on ice for 1 h. For the competitor control (comp)a 10-fold excess of non-biotinylated oligo was added. The negativecontrol (nc) contained cell lysate, herring sperm DNA and buffer H.Buffer H equilibrated streptavidin-agarose beads (Novagen™) were addedto pull-downs and controls and then incubated for 30 min at 4° C. on arotator. The beads were centrifuged, washed several times with buffer H,boiled in Laemmli buffer and separated by SDS-PAGE. C/EBPβ was detectedby Western blot (Santa Cruz Biotech™). 5′-biotinylated andnon-biotinylated oligos were ordered from MicroSynth: C/EBPβ_for: TATTAG CCA ATA TTA GCC AAT ATT AGC CAA TAT TAG CCA (SEQ ID NO: 21),C/EBPβ_rev: TGG CTA ATA TTG GCT AAT ATT GGC TAA TAT TGG CTA ATA (SEQ IDNO: 22).

Total RNA isolation, reverse transcription and quantitative reversetranscriptase-polymerase chain reaction (qRT-PCR): Cells werehomogenized and isolated with Trifast Reagent (PEQLAB BiotechnologyGmbH™) following the manufacturer's instruction. cDNAs were transcribedusing the High Capacity cDNA Reverse Transcription kit (Fermentas™) asindicated in the instruction manual. Expression of mRNA was quantifiedby real time PCR using Fast SYBR Green Master Mix (Applied Biosystems™)with the StepOne Real-Time PCR System (Applied Biosystems™) and primersin TABLE 3. Samples were assayed in duplicates dependent on the qualityof their melting curves. Levels of target genes were normalized to HPRTor GAPDH and described as fold induction of unstimulated cells.

TABLE 3 Target Forward (5′to 3′) SEQ ID NO: Reverse (5′to 3′) SEQ ID NO:PTEN ACA CCG CCA 23 TAC ACC AGT 28 AAT TTA ACT GC CCG TCC CTT TCArginase GTG AAG AAC 24 CTG GTT GTC 29 1 CCA CGG TCT GT AGG GGA GTG TTYm-1 TTT CTC CAG TGT 25 TCT GGG TAC 30 AGC CAT CCT T AAG ATC CCT GAAFizz-1 CTG GAT TGG 26 CCC TTC TCA 31 CAA GAA GTT CC TCT GCA TCT CCStabilin-1 CCC TCC TTC TGC 27 CAA ACT TGG 32 TCT GTG TC TGT GGA TGT CG

Enzyme-linked immunoabsorbent assay (ELISA):Supernatant levels ofselected cytokines secreted by lymphocytes or macrophages were measuredby utilizing commercially available enzyme-linked immunosorbent assay(ELISA) (all from eBioscience) for the quantification of IL-2, IFN-γ,IL-6, IL-12/23 (p40/common subunit), and IL-17A according to themanufacturer's protocol. IL-6 and IL-12/23 were analyzed in thesupernatants of macrophages 24 h after stimulation with LPS. IL-2, IFN-γand IL-17A were measured in supernatants collected at day 4 ofallogeneic mixed leucocyte reactions and of re-stimulated splenocytesand lymphocytes of Myelin oligodendrocyte glycoprotein (MOG)-immunizedmice. Briefly, plates were coated with capture antibody, blocked, anddiluted samples and standards were loaded for overnight incubation.Next, plates were incubated with detection antibody and then withStreptavidin-HRP (R&D Systems™) for development TMB 2—ComponentMicrowell Peroxidase.

Induction of experimental autoimmune encephalomyelitis (EAE) and ex-vivorestimulation: Mice were assigned to 4 groups each consisting of 4 to 8mice (see study design in TABLE 4). Briefly, for immunization mice wereinjected with 150 μl of an emulsion containing equal parts of MOG₃₅₋₅₅(1 mg/ml, Charite Berlin™) and IFA (Sigma™) supplemented with 10 mg/mlMycobacterium tuberculosis H37Ra (Difco™).

At time of immunization and second day after immunization 200 ngPertussis toxin (Calbiochem™) were administered by the intraperitonealroute. For in vivo administration of purified recombinant human ArginaseI mice were intravenously injected with either 10 mg/kg of body weightat days −4 and −2 prior immunization, or with 10 mg/kg or 1 mg/kg ofbody weight at days −4, −2 pre-, and 5 and 7 post-immunization. Micewere observed daily for clinical signs. Progression of EAE was dividedin 4 clinical stages: grade 0: no signs, grade 1: complete floppy tail,grade 2: severe paraparesis, grade 3: tetraparesis, grade 4: moribundcondition. For ex vivo stimulation lymphocytes and splenocytes wereisolated and stimulated with 30 μg/ml of the cognate MOG-peptide for 3days for proliferation analysis and cytokine measurements.

Mixed leucocyte reaction (MLR): Wildtype DCs were generated asdescribed. One part of the cells was stimulated with 30 μg/ml purifiedrecombinant human Arginase I (SEQ ID NO: 9) for 24 hrs at day 6 ofdifferentiation, the other part acted as unstimulated control. MLR wasperformed on day 7 of differentiation in absence of purified recombinanthuman Arginase I (SEQ ID NO: 9), which was removed by accurate washing.Briefly all cells were activated and loaded with LPS (100 ng/ml,Invitrogen™) and Ovalbumin (50 μg/ml, Sigma™) for 4 hours, washed andco-cultivated with OT-II cells. Responder T cells were isolated fromspleens of OT-II mice via positive magnetic cell sorting using a panT-cell isolation kit (Miltenyi Biotec™), labeled with 7 μM CFSE/1×10⁷cells according to the manufacturer's instructions (Sigma™). DCs (100000 cells) were washed and plated on a 48-well along with responderT-cells (500 000 cells) in 500 μl total volume. On day 3 of theco-culture cells were harvested, fixed and subjected to intracellularstaining. For proliferation cells were harvested in TruCount tubes andanalyzed for CFSE dilution.

CD4* T cell phenotyping by flow cytometry: Cells were isolated from MLRin vitro cultures on day 3 and re-stimulated with PMA/ionomycin (Sigma™)together with Golgi-Stop (BD Biosciences™) and analyzed forintracellular cytokines. The following antibodies were used for cytokinestaining: Anti-mouse CD4—PerCP (clone RM4-5, BD Pharmingen™),CD25—PE-Cy7 (clone PC61.5), IL-2—eFlour® 450 (clone JES6-5H4),IL-10—Alexa Flour® 647 (clone JES5-16E3), IL-17A—PE-Cy7 (clone 17B7),IFNγ-PE (clone XMG1.2, all from eBioscience™). Cell acquisition and dataanalysis was performed on a LSR 2 flow cytometer (BD Biosciences™) andFlowJo™ software Version 10.0 (Treestar™).

Statistics: Statistical significance of data was calculated by use of anunpaired two-tailed Student's t-test. Two way ANOVA analyses were usedto analyze two groups over time. Statistical analysis was performedusing GraphPad Prism software (GraphPad™ Software, La Jolla, USA).Results are presented as the mean+/−standard deviation. P-values <0.05were considered statistically significant (p-values were expressed asfollows: * p<0.05, ** p<0.01, *** p<0.001).

Results.

To better characterize the biological crosstalk between Arginase Iexpression and the activation of immune cells the expression profile ofArginase I mRNA under different conditions was investigated. Weidentified increased levels of Arginase I mRNA in a genome wide screenof PTEN deficient dendritic cells and littermate derived control cells.FIG. 1 illustrates the upregulation of Arginase I by LPS in macrophages.In FIG. 1, Thioglycollate elicited peritoneal macrophages (tPMs) wereinduced by E. coli O111:B4 LPS for 8 h and 24 h. Arginase I expressionwas increased on mRNA level 8 hours after LPS stimulation (FIG. 1, PanelA) and on protein level within 24 hours post LPS induction (FIG. 1,Panel B). These time points were selected for further analysis ofLPS-mediated Arginase I expression in dependence of the PI3K/PTENsignaling pathway in macrophages.

High expression of Arginase I in PTEN deficient macrophages suggestedthat one or more components of the signal transduction pathways involvedin innate immunity could regulate, or be regulated, by Arginase Iexpression. FIG. 2 illustrates an increase in Arginase I expressionaccompanied by loss of PTEN. FIG. 2, panel A illustrates the markedupregulation of Arginase I mRNA in unstimulated, naïve PTEN deficientperitoneal macrophages. These experiments were performed in residentperitoneal macrophages and in sterile inflammation induced peritonealmacrophages (FIG. 2, panels B and C). In accordance with upregulatedArginase I mRNA expression, increased mRNA levels for stabilin 1 inunstimulated PTEN^(−/−) tPMs (FIG. 2, panel D), and reduced expressionof YM1 and FIZZ (FIG. 2, panels E and F) was observed. The increase inprotein expression of Arginase I in PTEN negative cells was alsoconfirmed.

To further evaluate the function of Arginase I in response to LPSinduced signaling events the expression levels of Arginase I on mRNA andprotein levels was analyzed 24 hours post LPS activation (FIG. 2, panelsH and I). Wildtype macrophages harvested from littermate control miceshowed slight upregulation of Arginase I. Notably the expression of theprominent M1 marker iNOS was also enhanced in LPS stimulated PTENdeficient macrophages.

The following experiments were conducted to further study the mechanismsof a broader immune-regulatory function of Arginase I in immunecells: 1) wildtype macrophages were stimulated with LPS; 2) stimulationwas inhibited with the fungal PI3K inhibitor wortmannin, which has beenreported to enhance cytokine synthesis upon LPS induction in vitro andin vivo (Guha, M., and N. Mackman. 2002. The phosphatidylinositol3-kinase-Akt pathway limits lipopolysaccharide activation of signalingpathways and expression of inflammatory mediators in human monocyticcells. J. Biol. Chem. 277: 32124-32132); Schabbauer, G. et al. 2004.PI3K-Akt pathway suppresses coagulation and inflammation in endotoxemicmice. Arterioscler. Thromb. Vasc. Biol. 24: 1963-1969); and with 3) theArginase inhibitor N-hydroxy-nor-L-arginine (L-nor-Arg). Inhibition ofArginase I significantly enhanced IL-6 production in macrophages (FIG.2, panel J). The effects of the Arginase-specific inhibitor on thediminished cytokine production of PTEN^(−/−) macrophages was alsoevaluated as compared to wildtype macrophages. Indeed a significant, butonly partial, restoration of the wildtype IL-6 production was found upontreatment with L-nor-Arg in PTEN deficient tPMs (FIG. 2, panel K).

The pharmacologic Arginase inhibition and the ablation of theanti-inflammatory phenotype in PTEN deficient Arginase overexpressingtPMs support the claim that Arginase I contributes to the PI3K mediatedmodulation of inflammatory responses.

PTEN deletion in macrophages upregulates the transcription factorC/EBPβ, which is crucial for Arginase I promoter activation. Toelucidate the molecular mechanism responsible for the PTEN-mediated generegulation of Arginase I, several potential candidate transcriptionfactors (TF) were analyzed. The most prominent regulator of Arginase Iis STAT 6, which is activated by IL-4 and/or IL-13 in macrophages(Gordon, S., and F. O. Martinez. 2010. Alternative activation ofmacrophages: mechanism and functions. Immunity 32: 593-604).Unstimulated tPMs, either PTEN deficient cells or wild type cellsderived from littermate control animals, did not show any overt changesin STAT6 total protein content or activated STAT6 as measured byphospho-specific STAT6 antibodies. Another candidate transcriptionfactor is C/EBPβ, which has been shown to contribute to patternrecognition receptor-mediated regulation of Arginase I (El Kasmi, K. C.et al. 2008. Toll-like receptor-induced arginase 1 in macrophagesthwarts effective immunity against intracellular pathogens. Nat.Immunol. 9: 1399-1406).

To characterize mechanisms of modulating Arginase I expression bymodulating upstream factors, the regulation of C/EBPβ by PTEN, andsubsequent constitutive upregulation of Arginase I by C/EBPβ wereanalyzed. FIG. 3 illustrates the function of C/EBPβ in PTEN deficientmacrophages. FIG. 3, panel A illustrates the upregulation of C/EBPβprotein in PTEN deficient tPMs. FIG. 3, panel B illustrates that thesame result was obtained for two isoforms of C/EBPβ, LAP and LAP*.Further induction of the transcription factor was observed within 8hours post LPS induction. However we could not identify an additionalupregulation of C/EBPβ by PTEN deletion upon TLR4 activation at least atthe time point we have evaluated (8 h). Since maximal Arginase Iexpression is observed 24 h after LPS induction, we cannot excludeC/EBPβ differential expression might occur earlier in activated PTENdeficient macrophages.

Due to the fact that C/EBPβ is a transcription factor acting on specificDNA elements, the binding of C/EBPβ to the Arginase enhancer elementcontaining a number of different TF consensus binding sites wasinvestigated. An avidin biotin coupled DNA (ABCD) binding assay was usedto evaluate C/EBPβ DNA binding properties using biotinylated oligosspanning the C/EBPβ consensus site within the Arginase enhancer 3.8 kbupstream of the transcription start site. The suitability of thisexperimental system was tested in HEK cells overexpressing C/EBPβ.Overexpressed C/EBPβ efficiently bound the oligo which could beprecipitated together with the transcription factor, in particular theLAP* isoform, bound to it. Next the effects of PTEN deficiency on C/EBPβDNA binding in macrophages was analyzed. First the presence of C/EBPβ inthe lysates used for the ABCD binding assays was verified. Equal inputfor the ABCD assay was determined by reproving against GAPDH (FIG. 3,panel C). Analysis of TF binding to the Arginase oligo suggests that inaddition to increased protein levels enhanced binding of C/EBPβoccurred. LAP* was identified as the dominant isoform binding to theArginase enhancer oligo (FIG. 3, panel D).

These data suggest a potential mechanism for PI3K/PTEN regulation ofArginase I expression via C/EBPβ.

Example 2. Extracellular Activity of Arginase I

To study the cellular site of action of Arginase I, supernatants (SN) ofperitoneal macrophages were analyzed. Unexpectedly, Arginase I wasreleased into the extracellular space. This is in contrast to Arginase Icounterpart iNOS.

To further evaluate the role of Arginase I in cytokine production andthe potential of antigen presenting cells to polarize T-cells, mediaconditioned to wildtype bone marrow derived GM-CSF differentiateddendritic cells was transferred and their inflammatory response to LPSwas analyzed. FIG. 4 illustrates that constitutive activation of PI3Kpromotes Arginase I expression and release into the extracellular space.Surprisingly, the data indicates a downregulation in the protein levelsof the T-cell polarizing cytokines IL-6 and IL-12 p40, the commonsubunit of IL-12 and IL-23 (further on denoted as IL-12/23), (FIG. 4,panel D). Next we attempted to mimic a physiological environment withhigh expression of Arginase I conditioned media from PTEN deficientmacrophages, using purified recombinant human Arginase I (recArgI). Inorder to do so, dendritic cells stimulated by LPS were pre-incubatedwith purified recombinant human Arginase I. Reduced expression levels ofIL-6 and IL-12/23 similar to the treatment with conditioned media wasdetected. The decrease in expression was more pronounced on IL-12/23expression as compared to IL-6 (FIG. 4, panel E).

To further characterize the biological crosstalk between Arginase Iexpression and PTEN expression the expression of Arginase I andIL-4/IL-13 in PTEN deficient cells was measured. Deficiency of PTENleads to highly increased expression levels of Arginase I, even inunstimulated peritoneal macrophages tPMs (FIG. 4, panel A). Thesefindings were surprising and unexpected. To quantify the results,macrophages derived from 5 wildtype littermates and PTEN^(−/−) animalswere analyzed. A greater than 10-fold increase in extracellular ArginaseI expression was detected in PTEN^(−/−) macrophages (FIG. 4, panels Band C). In addition, we evaluated Arginase I secretion in response toLPS and in parallel to combined activation by IL-4 and IL-13. In bothcases we observed increased secretion in PTEN deficient macrophages,which was also seen to a limited extent in wildtype cells (FIG. 4,panels B bottom).

The result indicates that Arginase I presence in the extracellularenvironment of macrophages might exhibit anti-inflammatory properties ina paracrine fashion.

Example 3. Free Arginase I Potently Inhibits T-Cell Polarization

To analyze the effects of recombinant human Arginase I on antigenpresentation and T-cell polarizing properties, bone marrow derivedGM-CSF differentiated wildtype dendritic cells were pre-conditioned withrecombinant Arginase I overnight before the cells were loaded withOvalbumin (Ova) and stimulated with LPS. To avoid potential effects onT-cells, recArgI was removed from the DCs by multiple washing stepsbefore cultivating them together with isolated OT-II T-cells in a ratio5:1. Treatment of dendritic cells with recombinant Arginase I in aconcentration of 30 μg/ml did not alter the surface expression ofprototypic DC activation markers CD80 and MHCII, as measured by flowcytometry.

The results of the MLR were evaluated after 3 days of co-culture of DCsand T-cells. FIG. 5 illustrates inhibition of T-cell polarization byArginase I. We observed a significant reduction of Th1 and Th17signature cytokines IFNγ (FIG. 5, panels A and B) and IL17A (FIG. 5,panels D and E) expressing CD4⁺ T-cells. We note that IL-17 producingcells were present, but in low numbers. Analyzes of T-cell cytokinesthat were secreted during the MLR after LPS/Ova priming revealsignificant differences in the release of IFNγ, whereas IL-17A was notsignificantly different (FIG. 5, panels C and F). Proliferation asmeasured by CFSE dilution in dividing T-cells was evaluated. However wedid not find significant changes in the presence of recombinant ArginaseI (FIG. 5, panels G and H). The data suggests that extracellularArginase I in the presence of antigen-presenting cells amelioratesT-cell priming, thereby reducing the capacity to polarize preferentiallyin Th1 cells.

To further corroborate this hypothesis, the ability of recombinantArginase I to inhibit T cell polarization in vivo in a clinicallyrelevant model for T-cell mediated autoimmune disease was characterized.

Example 4. Recombinant Arginase I in the Treatment of Multiple Sclerosis

Autoimmune diseases are characterized by a deregulated immune system.The experimental autoimmune encephalomyelitis (EAE) mouse model is anart recognized animal model of multiple sclerosis. To study the abilityof a recombinant Arginase I to module an immune response, the responseof a mouse model for multiple sclerosis treated with a purified Arginasewas characterized. The EAE mouse reflects some, but not all, features ofthe human autoimmune pathology (Lassmann, H., and H. J. van. 2011. Themolecular basis of neurodegeneration in multiple sclerosis. FEBS Lett.585: 3715-3723). EAE was induced in wildtype mice by immunization withMOG₃₅₋₅₅ peptide in CFA (Lassmann, H., and H. J. van. 2011. Themolecular basis of neurodegeneration in multiple sclerosis. FEBS Lett.585: 3715-3723). In addition pertussis toxin was administered.

The potential efficacy of recombinant Arginase I in ameliorating diseaseprogression was characterized as follows: to determine a time-frame oftreatment, while antigen presentation and T-cell polarization were stillongoing, Arginase was administered in two different concentrationsbefore and shortly after immunization as described in TABLE 4.

TABLE 4 days (pre- or post-immunization) −4 −2 0 1 2 3 4 5 6 7 8 9 10 1112 13 14 15 16 17 Control: No treatment Group 1: + + + + recArgI 10mg/kg Group 2: + + + + recArgI 1 mg/kg Group 3: + + recArgI pre 10 mg/kg

FIG. 6 illustrates results of a treatment of experimental autoimmuneencephalomyelitis (EAE) with recombinant human Arginase I. The controlgroup developed visible signs of EAE after 12 days, further increasinguntil day 17. Pretreatment with recombinant Arginase I (10 mg/kg) didnot have any effect on the course of disease (FIG. 6, panel C). Pre- andpost-treatment with recombinant Arginase I (10 mg/kg) in contrast wascharacterized by a significant reduction in the onset as well as themagnitude of the disease (FIG. 6, panel A). In the same experimentalsetup, a minimum therapeutically effective dose of a pegylated ArginaseI (SEQ ID NO: 9) was tested (1 mg/kg). Treatment delayed the onset ofdisease, but disease progression and severity was not significantlyinhibited by the treatment with the lower dose of the pegylated ArginaseI (FIG. 6, panel B).

To analyze the MOG-specific T-cell response on a molecular level weharvested spleens and the draining lymph node (inguinal) andrestimulated the splenocytes and the lymph node cells, derived fromrecombinant human Arginase-treated and control EAE mice, in vitro withthe MOG₃₅₋₅₅ peptide. Restimulation of cells without further activationby exogenous stimuli led to a marked increase in IFNγ and IL-17Asecretion into the supernatants (see control vs. control+MOG in FIG. 6,panels D-G). Interestingly we found highly significant differences forreduced IFNγ (FIG. 6, panels D and E) and IL-17A (FIG. 6, panels F andG) levels in both pre- and post-treatment groups (1 mg/kg as well as 10mg/kg recombinant Arginase I) in particular in the draining lymph node.These data suggest that recombinant Arginase I treatment in the phase ofantigen presentation at least at the higher concentration efficientlyblunts EAE pathology in mice through a diminished capacity of CD4⁺ Tcells to produce cytokines IFNγ and IL-17A indispensable for thedevelopment of EAE.

Example 5. Recombinant Arginase I in the Treatment of RheumatoidArthritis

Arthritis is an autoimmune condition associated with varied levels ofpain, swelling, joint stiffness and sometimes a constant ache around thejoint(s). There are over 100 different forms of arthritis, includingrheumatoid arthritis, psoriatic arthritis, and related autoimmunediseases. Septic arthritis is caused by joint infection.

The major complaint by individuals who have arthritis is joint pain.Pain is often a constant and may be localized to the joint affected. Thepain from arthritis is due to inflammation that occurs around the joint,damage to the joint from disease, daily wear and tear of joint, musclestrains caused by inflammation. To evaluate the role of a recombinanthuman Arginase I in the treatment of arthritis we measured severalclinical parameters of arthritic mice treated with the recombinantprotein. FIG. 7 depicts graphs measuring various clinical parameters ofarthritic mice treated with recombinant human Arginase I. FIG. 7, panelA illustrates the percentage variation in weigh of mice receiving eithersaline, 1 mg/kg/w or 10 mg/kg/w of recombinant human Arginase I. FIG. 7,panel B is a graph plotting the ca-collagen type II antibody titer(arbitrary units) of mice receiving the treatments previously described.FIG. 7, panels C and D are the measurements of two different clinicalparameters of the same mice described above, namely, paw swelling andgrip strength. The results suggest that weekly treatment of arthriticmice with recombinant human Arginase I ameliorate the disease but do notprevent disease progression. FIG. 8 is a photograph illustrating thereduction of paw swelling in arthritic mice with human recombinant humanArginase I. FIG. 8, panel A illustrates the swelling of an arthriticmice that received treatment with saline only. FIG. 8, panel Billustrates the swelling of an arthritic mice that received treatmentwith 10 mg/kg of pegylated human recombinant Arginase I. The statisticalquantitation of the data suggests that treatment of arthritic mice withboth 1 mg/kg and 10 mg/kg of recombinant human Arginase I reduces pawswelling, as measured by the decrease in wrist joint diameter.

FIG. 9 corresponds to graphs illustrating the quantitation of differentcytokines in arthritic mice. The animals in FIGS. 9 and 10 were treatedwith collagen immunization, which promoted arthritis in the mice. FIG. 9panels A and B indicate that only a modest decrease in the levels ofIL17A and IL12/23 is observed in the mice treated with recombinant humanArginase I. FIG. 9 panel C illustrates the reduction of systemic releaseof the inflammatory cytokine Interleukin 6 (IL-6) in arthritic micetreated with a recombinant human Arginase I. FIG. 10 panels A-C indicatethat the levels of pro-inflammatory cytokines are not changed incollagen induced arthritis (CIA) in DBA mice. As a comparison to thearthritis treatment, FIG. 11 illustrates the clinical score of a mousemodel of experimental autoimmune encephalomyelitis (EAE) treated withhuman recombinant human Arginase I (for further details, see Example 1).The results indicate that a purified recombinant human Arginase I caneffectively treat conditions of the immune system.

To test the effects of recombinant Arginase in an in vivo settinginvolving antigen presentation, cytokine release and T-cellpolarization, fluorescent-activated cell sorting experiments were usedto characterize populations of immune cells in EAE mice. Arginasetreatment was performed after the disease progressed to a phenotype ofpartial hind limb paralysis and indicated that treatment with at leastthe recombinant arginase of SEQ ID NO. 9 could promote faster recovery.FIG. 12 depicts the fluorescence-activated cell sorting analysis ofpopulations of immune cells in EAE mice treated with recombinant humanArginase I (FIG. 12, PANELS A-D). FIG. 13 depicts the results offluorescence-activated cell sorting experiments indicating thattreatment with recombinant human Arginase I prevents T-cellproliferation (FIG. 13, PANELS A-F).

Example 6. Pegylated Formulations of Recombinant Human Arginase I

In vitro and in vivo experiments point out the immune-modulatoryproperties of extracellular Arginase I. To formulate, evaluate, andoptimize pharmaceutical compositions for the administration ofrecombinant human Arginase I in vivo several experiments characterizingthe pegylation of recombinant Arginase I were conducted. Pegylation isthe process of covalent attachment of polyethylene glycol (PEG) polymerchains to another molecule. The covalent attachment of PEG to a drug ortherapeutic protein can reduce the immunogenicity and antigenicity ofthe recombinant Arginase I from the subject's immune system, andincrease the hydrodynamic size of the recombinant Arginase, which canprolong the half-life of a pegylated recombinant human Arginase I invivo.

FIG. 14 is a schematic of a process utilized for optimizing apharmaceutical composition comprising a recombinant human Arginase I.Three different methods of covalent attachment of different sizes of PEGmolecules to recombinant Arginase I molecules were tested. Forillustrative purposes, FIG. 14 describes the attachment of distinct PEGmolecules to SEQ ID NO: 1 by distinct methods, but it should beunderstood that the pegylation optimization strategy applies to any oneof SEQ ID NOS. 1-16. FIG. 14 illustrates three methods of covalentlyattaching a PEG polymer chains to, for example SEQ ID No. 1, namelyamine —NH₂ conjugation, cysteine (—SH) conjugation, and N-terminalmodification. The pegylation of various mutant recombinant humanArginases at specific residues was optimized as described in FIG. 14.

TABLE 5 summarizes the pegylation of various mutant recombinant humanArginases by cysteine (—SH) conjugation. FIG. 15 shows the levels ofserum Arginine depletion in Spragus Dawley rats obtained with thepegylated Arginases described in TABLE 5 with a single intravenous doseof 3 mg/kg.

TABLE 5 SEQ ID NO. MAL-PEG MW Pegylated Products Pegylation Site No. ofPEG SEQ ID NO: 2 20K M1-20K (45) Cys45 1 M1-20K (168) Cys168 1 M1-20K(45/168) Cys45 & Cys168 2 SEQ ID NO: 2 30K M1-30K (45) Cys45 1 M1-30K(168) Cys168 1 M1-30K (45/168) Cys45 & Cys168 2 SEQ ID NO: 5 40K M2-40K(45) Cys45 1 SEQ ID NO: 6 40K M3-40K (168) Cys168 1

The half-life T_(1/2), peak plasma concentration after drugadministration C_(max), and the integral of the concentration-time curveafter administration of a single dose AUC of pegylated SEQ ID NO. 2, SEQID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 9. —SH modified pegylatedarginases in six different rats is shown in TABLE 6. Each measurementwas obtained after administration of a single intravenous dose of 3mg/kg (mean±SD; n=6).

TABLE 6 Pegylated Products T_(½) (hours) C_(max) (μg/mL) AUC_(last) (h *μg/mL) AUG_(0-∞) (h * μg/mL) M1-20K (45/168) 28.8 ± 4.5 41.5 ± 9.51599.2 ± 183.8 1614.8 ± 188.8 M1-30K (45/168) 28.4 ± 8.4 32.4 ± 3.11113.6 ± 116.8 1192.9 ± 121.2 M2-40K (45) 27.5 ± 3.1 45.2 ± 5.1 2063.9 ±161.5 2080.5 ± 163.8 M3-40K (168) 15.1 ± 0.6  82.7 ± 32.0 524.6 ± 32.6541.1 ± 34.2 SEQ ID NO: 9 31.5 ± 5.3  48.9 ± 12.6 1626.2 ± 631.8 1675.6± 644.7

TABLE 7 summarizes the pegylation of various mutant recombinant humanArginases by amine (—NH₂) conjugation. FIG. 16 is a graph illustratingthe serum arginine depletion by recombinant human Arginase I withvarious arginases pegylated on Lys residues. FIG. 17 is a graphillustrating the degree of pegylation of various recombinant humanArginase I proteins by amine (—NH₂) conjugation.

TABLE 7 SPA-PEG Pegylation Pegylation Pegylation Ratio SEQ ID NO. MWProducts Sites (MALDI-Tof) SEQ ID NO: 5 5K M2-5K (Lys) Lys, NH₂ 6-12 SEQID NO: 6 5K M3-5K (Lys) Lys, NH₂ 6-12 SEQ ID NO: 8 5K M4-5K (Lys) Lys,NH₂ 6-12 SEQ ID NO: 7 5K M7-5K (Lys) Lys, NH₂ 6-12

The half-life T₂, peak plasma concentration after drug administrationC_(max), and the integral of the concentration-time curve afteradministration of a single dose AUC of various amine (—NH₂) modifiedpegylated arginases in six different rats is shown in TABLE 8. Eachmeasurement was obtained after administration of a single intravenousdose of 3 mg/kg (mean±SD; n=6). The pharmacokinetic data in ratsindicates that pegylation of recombinant human Arginase I at multiplesites with low molecular weight PEGs, such as methoxy poly(ethyleneglycol) succinimidyl proprionate (mPEG-SPA) can provide effectivearginine depletion in vivo.

TABLE 8 C_(max) AUC_(last) AUC_(INF) Pegylated Products T_(½) (hours)(μg/mL) (h * μg/mL) (h * μg/mL) SEQ ID NO: 1 (pegylated 43.8 ± 4.4  42.2± 3.6 1918.7 ± 271.0 2687.0 ± 290.2 with mPEG-SPA 5K) M2-5K(Lys) 40.7 ±13.2 36.0 ± 6.0 1585.0 ± 184.8 2505.6 ± 534.8 M3-5K(Lys) 53.5 ± 14.234.4 ± 7.4 2675.3 ± 560.4 3577.4 ± 865.8 M4-5K(Lys) 59.5 ± 9.9  34.7 ±8.7 2265.7 ± 659.8 3147.8 ± 673.0 M7-5K(Lys) 50.5 ± 13.0  56.5 ± 11.0 4574.6 ± 1268.2  4939.5 ± 1308.1 SEQ ID NO: 9 (pegylated 35.5 ± 2.6 63.2 ± 5.1 3430.3 ± 361.5 3578.5 ± 415.1 with mPEG-SPA 5K)

In addition, enzyme kinetic parameters were measured for various mutantArginases as described in TABLE 9. In sum, modification of recombinanthuman Arginases with small molecular weight PEGs does not result in areduction of enzymatic activity.

TABLE 9 Pegylated K_(m) V_(max) K_(cat) K_(cat)/K_(m) Products (mM)(μmol * mL⁻¹ * min⁻¹) (sec⁻¹) (mM⁻¹sec⁻¹) M1-20K (45/168) 1.76 0.0196286.5 162.4 M1-30K (45/168) 1.92 0.0249 363.1 188.8 M2-40K (45) 1.870.0252 367 196.1 M3-40K (168) 2.07 0.0263 383.1 185.1 M2-5K (Lys) 2.050.0254 370 180.8 M3-5K (Lys) 2.03 0.0345 503.2 246.9 M7-5K (Lys) 1.790.0314 459.2 255.2 SEQ ID NO: 9 2.12 0.0293 426.8 202.3

FIG. 18 is a graph illustrating the epitope analysis of a pegylatedrecombinant human Arginase I.

Example 7. Recombinant Arginase I in the Treatment of Human AutoimmuneConditions

As disclosed in previous examples, Arginase I functions as animmunemodulating protein on intra- and extra-cellular levels.Pharmacological interference with arginase mediated L-arginine depletioncan effectively ameliorate swelling, pain, and joint stiffness in artrecognized models of Multiple Sclerosis and Rheumatoid Arthritis(Examples 4 and 5).

Rheumatoid arthritis (RA) is characterized as a chronic, inflammatorydisease in which the immune system destroys synovial joints andaccessory structures. Due to the progressive nature of RA, thisautoimmune condition can cause extra-articular complications withinseveral organ systems. Administration of a recombinant Arginase I of thedisclosure can be used for the treatment of rheumatoid arthritis in ahuman.

Any one of the recombinant human Arginases disclosed in SEQ ID NOS: 1through 16 can be used for the treatment of an autoimmune condition in ahuman, such as multiple sclerosis or rheumatoid arthritis. The purifiedArginase can be pegylated. In some embodiments, the purified Arginase ispegylated via amine conjugation with a methoxy poly(ethylene glycol)succinimidyl proprionate (mPEG-SPA) oligomer that weighs about 5 kDa(Example 6). In other embodiments, the purified Arginase can bypegylated with any other suitable PEG oligomer.

Pegylation of the purified Arginase(s) can provide a pharmaceuticalcomposition for the treatment of RA that has low immunogenicity, forinstance, the pegylation of recombinant human Arginase I at multiplesites with low molecular weight PEGs, such as (5 kDa mPEG-SPA oligomers)effectively reduced the exposure of epitopes resulting in an effectivetreatment with low immunogenecity. Various PEG oligomers disclosedherein can be used to effectively reduce the exposure of epitopes of anArginase of the disclosure.

Example 8. Recombinant Arginase I in Organ Transplantation

Immune suppression dampens an abnormal immune response in autoimmunediseases but it can also reduce a normal immune response to preventrejection of transplanted organs or cells. Immunomodulator drugs areimportant in the management of organ transplantation. Any one of therecombinant human Arginases disclosed in SEQ ID NOs: 1-16 can be used asan immunomodulator in the management of organ transplantation. In someembodiments, the recombinant Arginase is pegylated. In some embodiments,the recombinant Arginase is pegylated via amine conjugation with amethoxy poly(ethylene glycol) succinimidyl proprionate (mPEG-SPA)oligomer that weighs about 5 kDa. In other embodiments, the purifiedArginase can by pegylated with any other suitable PEG oligomer.Pegylation of the recombinant Arginase can provide a pharmaceuticalcomposition for the treatment of RA that has low immunogenicity: thepegylation of recombinant human Arginase I at multiple sites with lowmolecular weight PEGs, such as (5 kDa mPEG-SPA oligomers) caneffectively reduce the exposure of epitopes resulting in the reductionof immunogenecity. Various PEG oligomers disclosed herein can be used toeffectively reduce the exposure of epitopes of an Arginase of thedisclosure.

Example 9. Treatment of Experimental Autoimmune Encephalomvelitis (EAE)with Recombinant Human Arginase I

The experimental autoimmune encephalomyelitis (EAE) mouse is an artrecognized model of multiple sclerosis. EAE is also widely used as ananimal model for T-cell-mediated autoimmune diseases.

To investigate the effectiveness of treating an inflammatory disease ina subject with a therapeutically-effective amount of a recombinantArginase, the effectiveness of a pegylated recombinant human Arginase I,SEQ ID NO: 9, in modulating the mRNA and protein expression ofInterferon gamma and IL-17A in stimulated T-cells was investigated(treatment protocol and T-cell isolation was as described in Example 1).FIG. 19 illustrates IFNγ and IL-17A mRNA expression levels from myelinoligodendrocyte glycoprotein (MOG) restimulated Tcells inhibited withpurified human Arginase I. FIG. 20 illustrates IFNγ and IL-17A proteinexpression levels from myelin oligodendrocyte glycoprotein (MOG)restimulated Tcells inhibited with purified human Arginase I. FIGS. 19and 20 illustrate a significant reduction in mRNA and protein levels ofIFNγ and IL-17A in MOG stimulated Tcells.

FIG. 21 illustrates improvements in clinical scores of experimentalautoimmune encephalomyelitis (EAE) mice treated with recombinant humanArginase I. In this experiment, antigen-presenting cells were treatedwith pegylated recombinant human Arginase I, SEQ ID NO: 9, ex vivo andtransplanted back into EAE mice. FIG. 21, Panel A, illustrates theclinical score of EAE mice that were not challenged with CFA andpertussis prior to receiving ex vivo stimulated antigen presentingcells. FIG. 21, Panel A, illustrates a delayed improvement in theclinical score of the mice that had not been challenged with CFA andpertussis.

FIG. 21, Panel B, illustrates the clinical score of EAE mice that werechallenged with CFA and pertussis prior to receiving ex vivo stimulatedantigen presenting cells. FIG. 21, Panel B, illustrates a more rapidclinical score improvement.

Example 10. Modulation of Osteoclast Differentiation with RecombinantHuman Arginase I

In healthy bone, bone formation and bone resorption are processesinvolved in the normal remodeling of bone. In the process of remodeling,cells called osteoclasts resorb bone tissues whereas cells calledosteoblasts deposit new bone tissue. Osteoclasts are important in theremodeling, maintenance, and repair of bones of the vertebral skeleton.For instance, osteoclast dysfunction has been associated withosteoporosis.

An osteoclast differentiation assay was employed to assess the abilityof the recombinant human Arginase disclosed in SEQ ID NO: 9 to modulateosteoclast differentiation. FIG. 22 is an schematic of an osteoclastdifferentiation assay. On day 0 of the differentiation assay, bonemarrow cells were isolated from male mice with standard techniques,i.e., bone marrow cells were flushed with PBS from the fibia, plated in10 cm tissue culture dishes and treated with 100 ng/ml of M-CSF. After 3days of differentiation induced by the M-CSF, cells were harvested andplated at a density of 100,000 cells/ml. The plated cells weresubsequently treated with 50 ng/ml RANKL and 30 ng/ml M-CSF. At days 7-8of the differentiation protocol osteoclasts were TRAP-stained andcounted. FIG. 23, Panel A is a graph enumerating the number ofosteoclasts with at least three nuclei that were counted in anosteoclast differentiation assay. As shown in FIG. 23, Panel A, bonemarrow cells were treated with recombinant Arginase I on days 3 and 6 ofthe differentiation protocol and together with RANKL incubation. FIG.23, Panel B is a representative microscopy picture illustratingTartrate-resistant acid phosphatase (TRAP) staining of cells in controldish (untreated) and cells that were treated with 300 ng/ml of therecombinant human Arginase of SEQ ID NOs: 9. The TRAP staining is amarker for osteoclasts. The data is presented as mean±SEM and n=two miceper group, and corresponds to two independent experiments. Threetechnical replicates were performed in each independent experiment. *represent p<0.05, *** represent p<0.001. B.

The expression levels of endogenous Arginase I mRNA were monitored byqPCR at different time points during the differentiation protocol. FIG.24 is a graph illustrating the expression levels of Arginase I incontrol cells on days 3 and 7 of the differentiation protocol ascompared to the levels of the housekeeping control gene HPRT. Thesequence of the Arginase primers used is disclosed in TABLE 3. FIG. 24illustrates that expression of Arginase I is lost duringosteoclastogenesis. Messenger RNA levels of Arginase I decrease afteraddition of RANKL on day 3 of osteoclast differentiation. The data ofFIG. 24 is presented as mean±SEM and n=5 mice per group, and are twocombined independent experiments. * Represents p<0.05.

To assess the ability of recombinant Arginase I to modulate osteoclastdifferentiation, increasing dosages of the recombinant human Arginase ofSEQ ID NOs: 9 were added to different petri dishes on days 3 and day 6of the differentiation protocol. The experimental protocol used is asfollows (a minimum of three different petri dishes received eachtreatment): a) a control dish was not treated with recombinant humanArginase; b) a first experimental dish was treated with a dose of 1ng/ml of the recombinant human Arginase on days 3 and day 6 of thedifferentiation protocol; c) a second experimental dish was treated witha dose of 10 ng/ml of the recombinant human Arginase on days 3 and day 6of the differentiation protocol; d) a third experimental dish wastreated with a dose of 100 ng/ml of the recombinant human Arginase ondays 3 and day 6 of the differentiation protocol; e) a fifthexperimental dish was treated with a dose of 1,000 ng/ml (1 μg/ml) ofthe recombinant human Arginase on days 3 and day 6 of thedifferentiation protocol. FIG. 25 illustrates that addition ofrecombinant human Arginase I (SEQ ID NO. 9) during days 3 and 6 ofosteoclast differentiation can modulate osteoclast formation. FIG. 25,Panel A shows that addition of 1,000 ng/ml of recombinant human ArginaseI (SEQ ID NO. 9) inhibited osteoclast formation. FIG. 25, Panel Billustrates representative microscope pictures of the cells treated withthe dosages of the recombinant human Arginase I (SEQ ID NO. 9) describedin a)-e). A recombinant human Arginase I (SEQ ID NO. 9) can blockosteoclastogenesis in a dose dependent manner at concentrations between100 and 300 ng/ml. FIG. 25, Panel B illustrates that cells incubatedwith 1,000 ng/ml appear morphologically as TRAP negative macrophagesdespite of incubation with RANKL. The data in FIG. 25 is presented asmean±SEM and n=6 mice per group, and are three combined independentexperiments, *** represents p<0.001.

To assess the direct effects of the recombinant human Arginase I (SEQ IDNO. 9) in modulating osteoclast differentiation, the differentiationprotocol described above was performed with the addition of untreated,denatured, and enzymatically inactivated recombinant human Arginase I(SEQ ID NO. 9). The assay assessed the ability of denatured andenzymatically inactivated Arginase I in promoting or inhibitingosteoclast formation. The Arginase I enzyme was either a) heat denatured(70° C. 10 min) or b) enzymatically inactivated with 300 μM of theinhibitor nor-NOHA. The Arginases were then added to separate dishes onday 3 of the differentiation protocol. FIG. 26 illustrates the number ofdifferentiated osteoclasts that could be enumerated in a differentiationassay supplemented with denatured and N(omega)-hydroxy-nor-arginine(nor-NOHA) treated recombinant human Arginase I (SEQ ID NO. 9) ascompared to a control, untreated dish. Normal osteoclast counts wereobserved, which suggests that Arginine availability is a prerequisitefor osteoclast formation. The data in FIG. 26 is presented as mean±SEMand n=2 mice per group, and are representative of one experiment. FIG.26 demonstrates that blockage of osteoclastogenesis is dependent on thecatalytic functions of Arginase I.

Osteoclasts are formed by the fusion of many cells derived fromcirculating monocytes in the blood, which are derived from hematopoieticstem cells. To assess the role of Arginase I in the differentiation ofhematopoietic stem cells, the differentiation protocol described abovewas performed with the addition of 1 μg/ml of recombinant human ArginaseI on day 0. FIG. 27 demonstrates that the addition of a 1 μg/ml dosageof recombinant human Arginase I recombinant Arginase I to hematopoieticstem cells on day 0 of the differentiation protocol does not influenceosteoclast formation. The data in FIG. 27 is presented as mean±SEM andn=4 mice per group. The data shows a combination of two independentexperiments.

To assess the effects of 10 μg/ml dosages of recombinant human ArginaseI in hematopoietic stem cells, the differentiation protocol describedabove was performed with the addition of 10 μg/ml of recombinant humanArginase I on days 0 and on day 6. FIG. 28 illustrates that addition ofa 10 μg/ml dosage of recombinant human Arginase I on hematopoietic stemcells (day 0 of differentiation; “d0”) can interfere with osteoclastformation. In addition, the addition of 1 μg/ml dosage of recombinanthuman Arginase I on cells that have been differentiated for 6 days (day6 of differentiation; “d6”) can still interfere with osteoclastformation. The data in FIG. 28 is presented as mean±SEM and n=2 mice pergroup, and are representative of one experiment.

To assess the effects of different dosages of recombinant human ArginaseI in the expression levels of genes involved in osteoclastogenesis, mRNAlevels of relevant genes of osteoclastogenesis were measured after 7days of differentiation (with and without incubation with recombinanthuman Arginase I). FIG. 29 illustrates the changes in expression levelsof the TRAP, RANK, NFAT c1, and c-FOS genes during differentiation withand without recombinant human Arginase I. Messenger RNA levels of RANK,NFATc1 and c-FOS are increased after addition of 100 ng/ml recombinanthuman Arginase I, indicating a stimulating effect of lower Arginase Iconcentrations. Addition of 1,000 ng/ml recombinant human Arginase Icorrelates with a strong downregulation of the TRAP, RANK, NFAT c1, andc-FOS genes genes. The data in FIG. 29 is presented as mean±SEM and n=4mice per group, and are two combined independent experiments.

Example 11. In-Vivo Modulation of Bone Conditions with Recombinant HumanArginase I

A mouse model of osteoporosis was created by removing the ovaries of 10week-old female mice. The procedure induced artificial menopause andbone loss in the mice and provided an in-vivo model for the study ofosteoporosis. Up to 30 mg/kg of the recombinant human Arginasesdisclosed in SEQ ID NOs: 9 was administered to the mice twice weekly.Mice were sacrificed and evaluated at 4 weeks after the start oftreatment. A histological analysis of the tibiae was performed to assessbone volume and destruction. The presence of osteoblasts and osteoclastsin this disease model were assessed using OsteoMeasure software(OsteoMetrics Inc., Atlanta).

EMBODIMENTS Embodiment 1

A method of treating an inflammatory disease in a subject in needthereof, the method comprising administering to the subject atherapeutically-effective amount of a purified Arginase, or a functionalfragment thereof.

Embodiment 2

The method of Embodiment 1, wherein the inflammatory disease isrheumatoid arthritis.

Embodiment 3

The method of Embodiment 1, wherein the inflammatory disease is multiplesclerosis.

Embodiment 4

The method of any one of Embodiments 1-3, wherein the purified Arginaseis recombinant Arginase.

Embodiment 5

The method of any one of Embodiments 1-4, wherein the recombinantArginase is pegylated.

Embodiment 6

The method of any one of Embodiments 1-5, wherein the pegylatedrecombinant Arginase is recombinant human Arginase I.

Embodiment 7

The method of any one of Embodiments 1-6, wherein the purified Arginaseis SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, or SEQ ID NO: 16.

Embodiment 8

The method of any one of Embodiments 1-7, wherein thetherapeutically-effective amount of the purified Arginase is from about1 mg/Kg to about 10 mg/Kg.

Embodiment 9

The method of any one of Embodiments 1-8, wherein thetherapeutically-effective amount of the purified Arginase is from about10 mg/Kg to about 100 mg/Kg.

Embodiment 10

The method of any one of Embodiments 1-9, wherein thetherapeutically-effective amount of the purified Arginase is greaterthan 100 mg/Kg.

Embodiment 11

The method of any one of Embodiments 1-10, wherein the purified Arginaseprovides an arginine plasma concentration in the subject that is lowerthan 120 μM.

Embodiment 12

The method of any one of Embodiments 1-11, wherein the purified Arginaseprovides an arginine plasma concentration in the subject that is lowerthan 80 μM.

Embodiment 13

The method of any one of Embodiments 1-12, wherein the purified Arginaseprovides an arginine plasma concentration in the subject that is lowerthan 10 μM.

Embodiment 14

The method of any one of Embodiments 1-13, wherein the administration isintravenous administration.

Embodiment 15

The method of any one of Embodiments 1-14, wherein thetherapeutically-effective amount of a purified recombinant arginase isin a unit dosage form.

Embodiment 16

The method of any one of Embodiments 1-15, wherein the subject is ahuman.

Embodiment 17

The method of any one of Embodiments 1-16, wherein the Arginase ispartially purified.

Embodiment 18

The method of any one of Embodiments 1-16, wherein the Arginase issubstantially pure.

Embodiment 19

The method of any one of Embodiments 1-16, wherein the Arginase is atleast 95% pure.

Embodiment 20

The method of Embodiment 19, wherein the Arginase is at least 99% pure.

Embodiment 21

A method of modulating inflammation, the method comprising administeringto a subject a therapeutically-effective amount of a purified Arginase,or a functional fragment thereof, wherein the administration modulatesthe inflammation.

Embodiment 22

The method of Embodiment 21, wherein the purified Arginase is arecombinant Arginase.

Embodiment 23

The method of any one of Embodiments 21 and 22, wherein the recombinantArginase is pegylated.

Embodiment 24

The method of any one of Embodiments 21-23, wherein the pegylatedrecombinant Arginase is pegylated recombinant human Arginase I.

Embodiment 25

The method of any one of Embodiments 21-24, wherein the purifiedArginase inhibits T-cell polarization.

Embodiment 26

The method of any one of Embodiments 21-25, wherein the purifiedArginase modulates cytokine release.

Embodiment 27

The method of any one of Embodiments 21-26, wherein the cytokine isInterleukin 6.

Embodiment 28

The method of any one of Embodiments 21-26, wherein the cytokine isInterferon gamma.

Embodiment 29

The method of any one of Embodiments 21-28, wherein the purifiedArginase I comprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ IDNo. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ IDNo. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQID No. 14, SEQ ID No. 15, or SEQ ID No. 16.

Embodiment 30

The method of any one of Embodiments 21-29, wherein the inflammation isassociated with an autoimmune disorder.

Embodiment 31

The method of Embodiment 30, wherein the autoimmune disorder is multiplesclerosis.

Embodiment 32

The method of Embodiment 30, wherein the autoimmune disorder isrheumatoid arthritis.

Embodiment 33

The method of any one of Embodiments 21-32, wherein the administrationof the purified Arginase provides a plasma level of arginine in thesubject that is no greater than 10 μM.

Embodiment 34

The method of any one of Embodiments 31 or 32, wherein thetherapeutically-effective amount of the purified Arginase is from about1 mg/kg to about 10 mg/kg of the subject's body mass.

Embodiment 35

The method of any one of Embodiments 31 or 32, wherein thetherapeutically-effective amount of the purified Arginase is from about10 mg/kg to about 100 mg/kg of the subject's body mass.

Embodiment 36

The method of any one of Embodiments 31 or 32, wherein thetherapeutically-effective amount of the purified Arginase is greaterthan 100 mg/kg of the subject's body mass.

Embodiment 37

The method of any one of Embodiments 21-36, wherein thetherapeutically-effective amount of the purified Arginase isadministered to the subject at least once over a period of 24 hours.

Embodiment 38

The method of any one of Embodiments 21-37, wherein thetherapeutically-effective amount of the purified Arginase isadministered to the subject at least once over a period of 48 hours.

Embodiment 39

The method of any one of Embodiments 21-38, wherein thetherapeutically-effective amount of the purified Arginase isadministered to the subject at least once over a period of 1 week.

Embodiment 40

The method of any one of Embodiments 21-39, wherein thetherapeutically-effective amount of the purified Arginase isadministered to the subject at least once over a period of 2 weeks.

Embodiment 41

The method of any one of Embodiments 21-40, wherein the subject is ahuman.

Embodiment 42

A method of modulating an immune response, the method comprisingadministering to a subject a therapeutically-effective amount of apurified Arginase, or a functional fragment thereof, wherein theadministration modulates the immune response.

Embodiment 43

The method of Embodiment 42, wherein the purified Arginase is arecombinant Arginase.

Embodiment 44

The method of Embodiment 43, wherein the recombinant Arginase ispegylated.

Embodiment 45

The method of Embodiments 44, wherein the pegylated recombinant Arginaseis pegylated recombinant human Arginase I.

Embodiment 46

The method of any one of Embodiments 42-45, wherein the purifiedArginase comprises SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No.4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9,SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No.14, SEQ ID No. 15, or SEQ ID No. 16.

Embodiment 47

The method of any one of Embodiments 42-46, wherein the modulating theimmune response suppresses the immune system of a subject

Embodiment 48

The method of Embodiment 47, wherein the suppression of the immunesystem of the subject facilitates a cell, a tissue, or an organtransplant into the subject.

Embodiment 49

A use of a purified recombinant arginase, or a functional fragmentthereof, in the preparation of a medicament for treating an inflammatorydisease in a subject.

Embodiment 50

The use of Embodiment 49, wherein the inflammatory disease is rheumatoidarthritis.

Embodiment 51

The use of Embodiment 49, wherein the inflammatory disease is multiplesclerosis.

Embodiment 52

The use of Embodiment 49, wherein the purified recombinant arginase is apegylated recombinant human Arginase I.

Embodiment 53

The use of Embodiment 52, wherein the medicament comprises less than1000 mg of the purified recombinant arginase.

Embodiment 54

The use of Embodiment 53, wherein the medicament comprises less than 100mg of the purified recombinant arginase.

Embodiment 55

The use of Embodiment 54, wherein the medicament comprises less than 10mg of the purified recombinant arginase.

Embodiment 56

The use of Embodiment 55, wherein the medicament comprises less than 1mg of the purified recombinant arginase.

Embodiment 57

The use of Embodiment 49, wherein the medicament provides an arginineplasma concentration in the subject that is lower than 120 μM.

Embodiment 58

The use of Embodiment 49, wherein the medicament provides an arginineplasma concentration in the subject that is lower than 80 μM.

Embodiment 59

The use of Embodiment 49, wherein the medicament provides an arginineplasma concentration in the subject that is lower than 10 μM.

Embodiment 60

The use of Embodiment 49, wherein the administration is intravenousadministration.

Embodiment 61

The use of Embodiment 49, wherein the subject is human.

Embodiment 62

A pharmaceutical composition comprising, a purified recombinant humanArginase I protein, or a functional fragment thereof, and at least onepolyethylene glycol oligomer.

Embodiment 63

The pharmaceutical composition of Embodiment 62, wherein the pegylatedrecombinant human Arginase I protein comprises at least two polyethyleneglycol oligomers.

Embodiment 64

The pharmaceutical composition of Embodiment 62, wherein eachpolyethylene glycol oligomer weighs from about 20 kilodaltons and about40 kilodaltons.

Embodiment 65

The pharmaceutical composition of Embodiment 63, wherein the pegylatedrecombinant human Arginase I protein comprises from about 4 polyethyleneglycol oligomers to about 13 polyethylene glycol molecules.

Embodiment 66

The pharmaceutical composition of Embodiment 62, wherein thepolyethylene glycol oligomer weighs about 5 kilodaltons.

Embodiment 67

The pharmaceutical composition of Embodiment 62, wherein thepolyethylene glycol oligomer is conjugated to a cysteine residue of thepurified recombinant human Arginase I.

Embodiment 68

The pharmaceutical composition of Embodiment 62, wherein thepolyethylene glycol oligomer is conjugated to an amine residue of thepurified recombinant human Arginase I protein.

Embodiment 69

The pharmaceutical composition of Embodiment 62, wherein thepolyethylene glycol oligomer is conjugated to the N-terminus of thepurified recombinant human Arginase I protein.

Embodiment 70

The pharmaceutical composition of Embodiment 62, wherein thepharmaceutical composition is packaged as a kit.

Embodiment 71

A method of treating a bone disease in a subject in need thereof, themethod comprising administering to the subject atherapeutically-effective amount of a purified Arginase, or a functionalfragment thereof.

Embodiment 72

The method of Embodiment 71, wherein the bone disease is osteoporosis.

Embodiment 73

The method of Embodiments 71 and 72, wherein the osteoporosis isassociated with an osteoclast dysfunction.

Embodiment 74

The method of any one of Embodiments 71-73, wherein the purifiedArginase is recombinant Arginase.

Embodiment 75

The method of any one of Embodiments 71-74, wherein the recombinantArginase is pegylated.

Embodiment 76

The method of any one of Embodiments 71-75, wherein the pegylatedrecombinant Arginase is recombinant human Arginase I.

Embodiment 77

The method of Embodiment 76, wherein the pegylated recombinant humanArginase comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, or SEQ ID NO: 16.

Embodiment 78

The method of any one of Embodiments 71-77, wherein thetherapeutically-effective amount of the purified Arginase is from about1 mg/Kg to about 10 mg/Kg.

Embodiment 79

The method of Embodiment 78, wherein the therapeutically-effectiveamount of the purified Arginase is from about 10 mg/Kg to about 100mg/Kg.

Embodiment 80

The method of Embodiment 79, wherein the therapeutically-effectiveamount of the purified Arginase is greater than 100 mg/Kg.

Embodiment 81

The method of any one of Embodiments 71-79, wherein the purifiedArginase provides an arginine plasma concentration in the subject thatis lower than 120 μM.

Embodiment 82

The method of Embodiment 81, wherein the purified Arginase provides anarginine plasma concentration in the subject that is lower than 80 μM.

Embodiment 83

The method of Embodiment 82, wherein the purified Arginase provides anarginine plasma concentration in the subject that is lower than 10 μM.

Embodiment 84

The method of any one of Embodiments 71-83, wherein the administrationis intravenous administration.

Embodiment 85

The method of any one of Embodiments 71-84, wherein thetherapeutically-effective amount of a purified recombinant arginase isin a unit dosage form.

Embodiment 86

The method of any one of Embodiments 71-85, wherein the subject is ahuman.

Embodiment 87

The method of any one of Embodiments 71-86, wherein the Arginase ispartially purified.

Embodiment 88

The method of any one of Embodiments 71-87, wherein the Arginase issubstantially pure.

Embodiment 89

The method of any one of Embodiments 71-88, wherein the Arginase is atleast 95% pure.

Embodiment 90

The method of Embodiment 89, wherein the Arginase is at least 99% pure.

What is claimed is:
 1. A method of treating rheumatoid arthritis in asubject in need thereof, the method comprising administering to thesubject suffering from rheumatoid arthritis a therapeutically-effectiveamount of a purified pegylated Arginase I or a functional fragmentthereof, wherein the purified pegylated Arginase I or functionalfragment thereof treats the rheumatoid arthritis by inhibiting T-cellpolarization.
 2. The method of claim 1, wherein the purified pegylatedArginase I is a purified pegylated recombinant Arginase I.
 3. The methodof claim 2, wherein the purified pegylated recombinant Arginase I is apurified pegylated recombinant human Arginase I.
 4. The method of claim1, wherein the therapeutically-effective amount of the purifiedpegylated Arginase I or the functional fragment thereof is from about 1mg/Kg to about 100 mg/Kg of the subject's weight.
 5. The method of claim1, wherein the therapeutically-effective amount of the purifiedpegylated Arginase I or the functional fragment thereof is about 10mg/Kg.
 6. The method of claim 1, wherein the purified pegylated ArginaseI or the functional fragment thereof provides an arginine plasmaconcentration in the subject that is lower than 120 μM.
 7. The method ofclaim 1, wherein the purified pegylated Arginase I or the functionalfragment thereof provides an arginine plasma concentration in thesubject that is lower than 10 μM.
 8. The method of claim 1, wherein theadministration is intravenous administration.
 9. The method of claim 1,wherein the subject is a human.
 10. The method of claim 1, wherein thepurified pegylated Arginase I or the functional fragment thereof is atleast 95% pure.
 11. The method of claim 1, wherein thetherapeutically-effective amount of the purified pegylated Arginase I orthe functional fragment thereof is administered to the subject at leastonce over a period of 24 hours.
 12. The method of claim 11, wherein thetherapeutically-effective amount of the purified pegylated Arginase I orthe functional fragment thereof is administered to the subject at leastonce over a period of 1 week.
 13. The method of claim 1, wherein thepurified pegylated Arginase I or the functional fragment thereofmodulates cytokine release in the subject.
 14. The method of claim 13,wherein the cytokine is Interleukin
 6. 15. The method of claim 13,wherein the cytokine is Interferon gamma.
 16. A method of treatingrheumatoid arthritis in a subject in need thereof, the method comprisingadministering to the subject a therapeutically-effective amount of apurified pegylated Arginase I or a functional fragment thereof, whereinthe purified pegylated Arginase I or the functional fragment thereofreduces a pain associated with rheumatoid arthritis in the subject. 17.The method of claim 16, wherein the purified pegylated Arginase I or thefunctional fragment thereof inhibits T-cell polarization.
 18. The methodof claim 16, wherein the pain is joint pain.
 19. The method of claim 16,wherein the purified pegylated Arginase I or the functional fragmentthereof reduces a swelling in the arthritic subject.
 20. The method ofclaim 16, wherein the subject is a human.
 21. A method of treatingrheumatoid arthritis in a subject in need thereof, the method comprisingadministering to the subject a composition comprising a purifiedpegylated human Arginase I, wherein the purified pegylated humanArginase I comprises an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, and SEQ ID NO:
 16. 22. The method of claim 21,wherein the purified pegylated human Arginase I comprises the amino acidsequence of SEQ ID NO:
 9. 23. The method of claim 21, wherein thepurified pegylated human Arginase I comprises the amino acid sequence ofSEQ ID NO:
 1. 24. The method of claim 21, wherein the purified pegylatedhuman Arginase I is pegylated via amine conjugation, cysteine (SH)conjugation, or N-terminal modification with a PEG oligomer.
 25. Themethod of claim 24, wherein the purified pegylated human Arginase I ispegylated via amine conjugation with a PEG oligomer.
 26. The method ofclaim 25, wherein the PEG oligomer weighs less than about 10 kDa. 27.The method of claim 25, wherein the PEG oligomer is methoxypoly(ethylene glycol) succinimidyl proprionate (mPEG-SPA).
 28. Themethod of claim 24, wherein the purified pegylated human Arginase I haslow immunogenicity.
 29. The method of claim 21, wherein the purifiedpegylated human Arginase I is expressed from bacterial cells or insectcells.
 30. The method of claim 1, wherein the rheumatoid arthritis isassociated with IL-17 inflammation.